Method of treating immune pathologies with low dose estrogen

ABSTRACT

The invention provides a method of ameliorating a Th1-mediated immune pathology in a mammal. The method is practiced by administering a low dose of estrogen to the mammal. Optionally, an immunotherapeutic agent can also be administered to the mammal. Also provided are kits containing a low dose of estrogen and an immunotherapeutic agent.

PRIORITY CLAIM

This is a § 371 U.S. national stage of International Application No.PCT/US01/40710, filed May 11, 2001, which was published in English underPCT Article 21(2), and claims the benefit of U.S. ProvisionalApplication No. 60/203,980 filed May 12, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the fields of immunology andmedicine and, more specifically, to the use of low dose estrogen totreat immune pathologies.

2. Background Information

The involvement of female sex hormones in immune pathologies has beenproposed based on a number of clinical and experimental observations.First, a variety of autoimmune diseases, including multiple sclerosis,rheumatoid arthritis and Grave's disease, preferentially affect women,and first occur during the reproductive years. Second, during pregnancy,when levels of female sex hormones are high, clinical remissions ofcell-mediated autoimmune diseases are common, with disease exacerbationoften seen post-partum when sex hormone levels are low. Third, in animalmodels of autoimmune disease, administration of estrogen at levels equalto or greater than those found in pregnancy has been shown to suppressthe clinical and histopathological symptoms of the disease. Fourth, invitro, estrogen at the high concentrations found in pregnancy has beenshown to inhibit production of inflammatory cytokines and to stimulateproduction of anti-inflammatory cytokines by autoantigen-specific CD4+cells from multiple sclerosis patients. However, in the same study, lowconcentrations of estrogen had the opposite effect, stimulatingproduction of inflammatory cytokines, with little or no effect onproduction of anti-inflammatory cytokines (Correale et al., J. Immunol.161:3365-3374 (1998).

To explain these observations, it has been proposed that the response toestrogen is biphasic, with high levels associated with protection fromautoimmune disease, and low levels associated with promotion of disease.However, because of the potential adverse effects of high levels ofestrogen on the reproductive and circulatory systems, and because of thepotential unwanted side effects in males, administration of high levelsof estrogen is unlikely to be widely useful as a therapy.

The effect of administering low dose estrogen to an individual with animmune disease has not previously been tested, although, from theclinical and experimental observations described above, little or nobeneficial effect on the course of the disease would be predicted.

There exists a need to design effective therapies that are applicablefor treating a variety of immune pathologies, in both genders, withminimal side effects. The present invention satisfies this need andprovides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a method of ameliorating a Th1-mediated immunepathology in a mammal. The method is practiced by administering a lowdose of estrogen to the mammal. Optionally, an immunotherapeutic agentcan also be administered to the mammal. Also provided are kitscontaining a low dose of estrogen and an immunotherapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of various concentrations of 17β-estradiol (E2)on the severity of EAE in female SJL mice.

FIG. 2 shows the effect of various concentrations of estriol (E3) on theseverity of EAE in female SJL mice.

FIG. 3 shows the effect of E2 and E3 on the severity of EAE in male SJLmice.

FIG. 4 shows the effect of low dose estrogen therapy on the PLP 139-151induced proliferation of draining lymph node (DLN) cells from female SJLmice with EAE.

FIG. 5 shows the effect of low dose estrogen therapy on PLP 139-151induced cytokine production by DLN cells from female SJL mice with EAE.

FIG. 6 shows the effect of estrogen therapy on PLP 139-151 specificimmunoglobulin production in female SJL mice with EAE.

FIG. 7 shows the effect of administration of BV8S2 (Vβ8.2) protein inIFA on the development of EAE in male and female mice.

FIG. 8 shows the effect of 17β-estradiol and estriol on cumulative EAEdisease index in intact and ovariectomized female mice.

FIG. 9 shows the effect of administration of BV8S2 protein, 17β-estrdioland the combination of BV8S2 vaccination plus estrus levels of17β-estradiol on EAE in Tg females.

FIG. 10 shows T cell proliferative (A-C) and antibody (D) responses inBV8S2 and/or E2 treated and control Tg mice challenged to develop EAE.

FIG. 11 shows the effect of ovariectomy (OVX) and treatment with estruslevels of 17β-estradiol (E2) on the course of EAE in Tg female mice.

FIG. 12 shows data obtained from RPA analysis of chemokine mRNAexpression in spinal cords of intact and ovariectomized 17β-estradioltreated and control TCR BV8S2 transgenic female mice.

FIG. 13 shows data obtained from RPA analysis of chemokine expression inspinal cord (SC) tissue and mononuclear cells from SC of BV8S2transgenic mice with EAE.

FIG. 14 shows data obtained from RPA analysis of chemokine receptorexpression in SC of 17β-estradiol treated, ovariectomized and controlTCR BV8S2 transgenic female mice with EAE.

FIG. 15 shows data obtained from RPA analysis of cytokine mRNAexpression in SC of 17β-estradiol treated and control TCR BV8S2transgenic female mice with EAE .

FIG. 16 shows the effect of 17β-estradiol on in vitro proliferative (A)and cytokine (B) responses of lymphokine (LN) T cells from naïve TCRBV8S2 transgenic female mice.

FIG. 17 shows the effect of 17β-estrodiol on the severity of EAE inwildtype C57BL/6 and cytokine knockout mice.

FIG. 18 shows data obtained from RPA analysis of chemokine and chemokinereceptor mRNA expression in the spinal cords of untreated and estrogentreated wildtype and cytokine knockout mice with EAE.

FIG. 19 shows cytokine production in the CNS of untreated and estrogentreated wildtype and cytokine knockout mice. Panel A shows data obtainedfrom RPA analysis of total RNA from the spinal cords of mice at the peakof EAE. Panel B shows by FACS analysis the percentages of Vβ8.2+, MOG35-55 stimulated T cells that express the indicated cytokines with orwithout 17β-estradiol treatment.

FIG. 20 shows the effect of estrogen treatment on MOG 35-55 stimulated Tcell proliferation and the expression of cell adhesion and activationmarkers in wildtype and cytokine knockout mice.

FIG. 21 shows the effect of estrogen treatment on the frequency of Vβ8.2T cells expressing the indicated cytokines.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed herein, administration of a low dose of estrogenunexpectedly reduces the severity of Th1-mediated immune pathologies.Additionally, low dose estrogen and immunotherapeutic agents actsynergistically in reducing the severity of Th1-mediated immunepathologies. These effects appear to be due, in part, to the effect oflow dose estrogen on reducing the expression of pro-inflammatorycytokines and chemokines by T cells in the periphery and at the site ofthe pathology.

Therefore, the invention provides a novel method of preventing orameliorating immune pathologies in a mammal by administering to themammal a low dose of estrogen. Optionally, the method further comprisesadministering an immunotherapeutic agent. The methods are advantageousin that low doses of estrogen can be administered to both males andfemales to prevent or ameliorate immune pathologies. Side effects ofhigh dose estrogen therapy, which are detrimental in females andpreclude its use in males, are expected to significantly reduced byadministering low dose estrogen. Additionally, the synergistic effectsof low-dose estrogen and an immunotherapeutic agent in inhibitingpathogenic immune responses allow a lower dose of agent to be used thanrequired using the agent alone, which reduces potential side effects andlowers the cost of therapy.

The methods of the invention can be practiced with respect to a varietyof immune pathologies. As used herein, the term “immune pathology”refers to a pathology mediated by a detrimental immune response. Suchpathologies are known in the art and include, but are not limited to,autoimmune pathologies, immune reactions by or against allografts,immune responses to infectious agents, chronic immune responses,allergic reactions and immunosuppressive responses andimmunoproliferative pathologies. Prognostic indicators and clinicalsigns associated with particular immune pathologies are well known inthe art. Additionally, as described below, immunotherapeutic agentsuseful in preventing or treating immune pathologies are well known inthe art, which can effectively be used in combination with low doseestrogen therapy in the methods of the invention.

Those skilled in the art appreciate that many immune pathologies aremediated by a combination of Th1, Th2, antibody, B cell, phagocytic andcomplement responses. Preferably, the methods of the invention arepracticed with respect to “Th1-mediated pathologies,” which arepathologies in which the detrimental immune response is primarily orpartially a T helper 1 (Th1) type immune response. A Th1 immune responseis characterized by secretion of pro-inflammatory cytokines, whichinclude IL-12, IFN-γ and TNF-α. Th1-mediated pathologies include mostautoimmune diseases, many alloimmune disorders, certain allergicconditions, certain chronic inflammatory conditions, and certaininfectious conditions.

An immune pathology can alternatively be mediated by a “Th2 immuneresponse,” which indicates that the detrimental immune response ispartially or primarily of the T helper 2 (Th2) type. A Th2 response ischaracterized by secretion of anti-inflammatory cytokines, such as IL-4,IL-10, IL-13 and TGF-β.

In most autoimmune pathologies, T cells recognize a host component inone or more tissues as foreign, and attack that tissue. Exemplaryautoimmune pathologies affecting mammals include rheumatoid arthritis(RA), juvenile oligoarthritis, collagen-induced arthritis, Sjogren'ssyndrome, multiple sclerosis (MS), experimental autoimmuneencephalomyelitis (EAE), inflammatory bowel disease (e.g. Crohn'sdisease, ulceritive colitis), autoimmune gastric atrophy, pemphigusvulgaris, psoriasis, vitiligo, type I diabetes, myasthenia gravis,Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis,sclerosing sialadenitis, systemic lupus erythematosis, Addison'sdisease, systemic sclerosis, polymyositis, dermatomyositis, perniciousanemia, and the like.

Alloimmune pathologies occur when tissue is transplanted from a donorwhose HLA antigens do not completely match the recipient antigens. Thedonor cells can be recognized by the recipient immune system as foreign,resulting in rejection of the transplanted tissue. Alternatively, donorimmune cells can recognize the recipient tissues as foreign, and attackthe recipient (graft versus host disease). Thus, a pathogenic alloimmuneresponse can be a response by or against a transplanted organ (e.g.heart, blood vessel, valve, liver, lung, kidney, skin) or infusedhematopoietic cells, such as an apheresis product or bone marrow.

Septic shock is a life-threatening response to infectious agents. Highlevels of bacterial toxins, including exotoxins and endotoxins, caninappropriately activate the host immune system to producehypersensitivity reactions that rapidly leading to septic shock withassociated organ failure and death. Septic shock is associated with bothgram-negative bacteria, such as Staphylococcus species, Streptococcusspecies, as well as gram-positive bacteria, and often is associated withinfection following surgery or trauma.

Chronic inflammatory responses are often initially responses toinfectious agents, but can be of any etiology, including tissue trauma.Chronic inflammatory responses are associated with a variety ofdiseases, including cardiovascular disease, coronary disease, cirrhosis,arthritis, cholestasis, tuberculosis, leprosy, syphilis, periodontitis,fibrosis, glomerulonephritis, and certain cancers.

Infectious agents that cause chronic inflammatory immune responsesinclude, for example, bacteria (e.g. Helicobacteria; Mycobacteria;Spirochocae; Yersinia and the like), viruses (e.g. HIV, hepatitisviruses, herpes simplex viruses, papovavirus, rabies virus), fungi,protozoa, helminths and prions.

Allergic reactions are hypersensitivity reactions to agents in theenvironment. Allergic reactions can be IgE/mast cell mediated, antibodymediated, Th1 or Th2 cell mediated, or a combination thereof. Allergicconditions and their etiology are well known in the art. Common allergicconditions include, for example, asthma, hay fever and food allergies.

Other immune pathologies that can be amenable to treatment with low doseestrogen include immune deficiency disorders, wherein a mammal mounts aninadequate immune response. Immune deficiencies can be caused, forexample, by HIV, the causative agent of AIDS; by malignancy; by old age;by malnutrition; by metabolic disease; by drug therapy; or bysplenectomy. Immune pathologies that can be amenable to treatment withlow dose estrogen include further include T cell replicativepathologies, including T cell leukemias and lymphomas.

Low dose estrogen therapy, alone or in combination withimmunotherapeutic agents or conventional therapies (e.g. antibiotics,antiviral agents, chemotherapy, radiation, as appropriate for theparticular disease), can be used to reduce the severity of the immunepathologies described above. As described herein, because of theinhibitory effect of low dose estrogen on TNFα expression, chemokineexpression and chemokine receptor expression, low dose estrogen therapywill be particularly useful in pathologies mediated by Th1 typeinflammatory responses.

As used herein, the term “ameliorating,” with reference to an immunepathology, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms in a susceptible mammal, a reduction in severity ofsome or all clinical symptoms of the disease, a slower progression ofthe disease, a reduction in the number of relapses of the disease, areduction in the number or activity (e.g. Th1 type cytokine secretion)of pathogenic T cells at the site of pathology or in the circulation, animprovement in the overall health or well-being of the individual, or byother parameters well known in the art that are specific to theparticular disease. Those skilled in the art can determine, based onknowledge of the expected course of the particular disease, whetherthere is a delayed onset of clinical symptoms. Those skilled in the artcan also determine whether there is an amelioration of the clinicalsymptoms or reduction in the number or activity of pathogenic T cellsfollowing treatment as compared with before treatment or as compared toan untreated mammal.

A useful method of monitoring the effect of a treatment that potentiallyameliorates multiple sclerosis is magnetic resonance imaging, or MRI. Asused herein, the term “magnetic resonance imaging” refers toconventional MRI methods, as well as improved magnetic resonance (MR)techniques, such as cell-specific imaging, magnetization transferimaging (MTI), gadolinium (Gd)-enhanced MRI, proton magnetic resonancespectroscopy (MRS), diffusion-weighted imaging (DWI), functional MRimaging (fMRI), and the other neuro-imaging methods known in the art.MRI methods and their applications to MS are described, for example, inRovaris et al, J. Neurol. Sci. 186 Suppl 1:S3-9 (2001). MRI techniquesallow an assessment of the effects of treatment on amelioration of avariety of well-known indicia of MS, including edema, blood brainbarrier break-down, demyelinisation, gliosis, cellular infiltration,axonal loss, T2 lesion load, T1 lesion load, gadolinium positive lesionload, and the like.

As used herein, the term “mammal” refers to a human, a non-humanprimate, canine, feline, bovine, ovine, porcine, murine or otherveterinary or laboratory mammal. Those skilled in the art understandthat the immune responses and immune pathologies of mammals share manycommon features, and that a therapy which reduces the severity of animmune pathology in one species of mammal is predictive of the effect ofthe therapy on another species of mammal. The skilled person alsoappreciates that credible animal models of many human immune pathologiesare known. As described in the Example, EAE is a credible animal modelof human multiple sclerosis.

As used herein, the term “estrogen” refers to the steroids commonlyknown as 17β-estradiol (E2), estrone (E1) and estriol (E3). Alsoincluded within the term “estrogen” are metabolites and derivatives ofE1, E2 and E3. Such metabolites and derivatives act as agonists of theestrogen receptor (ERα or ERβ) and have a similar core steroid structureas E1, E2 or E3, but can have one or more different groups (e.g.hydroxyl, ketone, halide, etc.) at one or more ring positions. Thoseskilled in the art can readily determine whether such metabolites andderivatives are agonists of estrogen by in vitro assays that measuresignaling through the estrogen receptor. Alternatively, the effects ofmetabolites and derivatives of estrogen can be assessed, and compared tothe effects of known estrogens, using any of the in vivo and in vitroassays that report estrogen's effects, as described in the Examples,below.

The methods of the invention can also be practiced with a non-steroidalestrogen analog that acts as an agonist of the estrogen receptor.Methods of identifying receptor agonists from libraries of compounds arewell known in the art, and include binding assays (e.g. competitive andnon-competitive radioimmunoassays) and signaling assays (e.g.transcription-based assays using reporter genes driven by an estrogenresponse element). Libraries of naturally occurring and syntheticcompounds, including inorganic compounds, peptides, lipids, saccharides,nucleic acids and small organic molecules, are commercially available,and can be screened in high-throughput assays to identify estrogenanalogs.

In the methods of the invention, estrogen is administered at a low butsufficient dose to reduce the severity of the particular immunepathology exhibited by the mammal. The dose will depend, among otherconsiderations, on the type of estrogen, its formulation and route ofadministration, the duration of therapy, the type and severity of thepathology, and on the weight and gender of the mammal.

As used herein, the term “low dose” refers to an amount sufficient toraise the serum concentration above basal levels, but below pregnancylevels. The diestrus, estrus and pregnancy serum concentrations of E2and E3 in mice are shown in Table 1. Human female physiologicconcentrations of E1 and E3 are roughly equivalent to those of E2, whichcirculates at 10 to 1,000 pg/ml during the normal menstrual cycle, andup to 35,000 pg/ml during pregnancy.

TABLE 1 17β-estradiol (pg/ml) Estriol (pg/ml) Diestrus 20-30 <100 Estrus100-200 <100 Pregnancy  5,000-10,000 2,000-3,000

Thus, a low dose of estrogen can raise serum E1, E2 or E3 to at least 10pg/ml, such as 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, 75 pg/ml, 100pg/ml, 150 pg/ml, 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 750 pg/ml,1000 pg/ml, 1500 pg/ml, and generally will not raise serum E1, E2 or E3beyond 2000 pg/ml. The amount of estrogen to administer to achievedesired hormone levels in the serum is known in the art, and willdepend, for example, on the weight of the mammal, the half-life of theparticular estrogen, and the route and form of administration. Theefficacy of a particular dose of estrogen can be monitored and adjustedduring therapy by examining standard disease parameters.

Those skilled in the art can determine an appropriate time and durationof therapy to achieve the desired preventative or ameliorative effectson the immune pathology. Thus, the methods of the invention can bepracticed so as to maintain low levels of estrogen in the blood forseveral days, weeks, months or years, or over the course of the lifetimeof the individual. For example, the therapy can be administeredcontinuously to an individual at risk of developing an immune pathology,such as an individual with a genetic predisposition to a pathology, orwith preclinical indications of the pathology. Likewise, estrogen can beadministered continuously to an individual early or late in the courseof the disease, or only administered during exacerbations of the diseaseuntil symptoms are controlled.

Low doses of estrogen can be prepared in any convenient form andadministered by any convenient route known in the art. Preferably, forhuman therapy, estrogen will be administered orally, transdermally,subcutaneously, intravenously, intramuscularly, by a respiratory route(e.g. inhalation), intranasal, enteral, topical, sublingual, or rectalmeans. Estrogen can also be administered directly to the site of thepathology, such as skin lesions, inflamed joints, into the centralnervous system, and the like. For continuous release of definedconcentrations of estrogen, administration via micropumps, biopolymers,liposomes and other slow-release vehicles is advantageous.

Optionally, low dose estrogen therapy can be combined withadministration of an immunotherapeutic agent. As used herein, the term“immunotherapeutic agent” refers to any compound used prophylacticallyor therapeutically to inhibit an immune response or to ameliorate animmune pathology. Preferably, the immunotherapeutic agent will beadministered at a lower dose than that required for complete efficacy onits own, such that when combined with administration of a low dose ofestrogen, there will be a pronounced effect on reduction of diseaseseverity not achieved by the immunotherapy alone. Administering a lowerdose of immunotherapeutic agent reduces the risk of adverse effects, aswell as reduces the cost of therapy.

The immunotherapeutic agent can be administered in combination withestrogen or separately; either before, at the same time, or afterestrogen administration; either by the same route or by a differentroute (e.g. any of the routes described above); and either at the samesite or at a different site. Those skilled in the art can determineappropriate conditions for administering both a low dose of estrogen andan immunotherapeutic agent to a mammal.

The choice of immunotherapeutic agent to use and route and site ofadministration will depend on the particular immune pathology. A varietyof agents with at least partial efficacy in treating immune pathologiesare known in the art, and their mechanisms of action are often wellunderstood. Other immunotherapeutic agents with similar mechanisms ofaction are in development.

The activation of a T cell immune response requires interaction betweena T cell receptor on the surface of the pathogenic T cell, and anantigenic peptide bound to an HLA (MHC) molecule on the surface of anantigen presenting cell or target cell. Any agent which disrupts thistrimolecular complex can be effective in combination with low doseestrogen therapy in reducing the T cell immune response. Agents whichdisrupt the trimolecular complex can be either immunomodulatory agentsor immunoblocking agents, or act by both an immunomodulatory and ablocking mechanism.

As used herein, the term “immunomodulatory agent” is intended to referto an agent that induces a host immune response, such as a tolerogenicresponse or an active immune response in a mammal.

Exemplary immunomodulatory agents that cause a tolerogenic response,which leads to immunological unresponsiveness, are autoantigens targetedby pathogenic T cells in an autoimmune response. Autoantigens and theadminstration of these autoantigens by a variety of routes (includingoral and intravenous routes) so as to induce tolerance are known in theart and described, for example, in U.S. Pat. Nos. 6,039,947; 6,019,971;5,869,093; 5,858,968 and 5,856,446.

Known or suspected autoantigens, with their associated diseases,include: myelin basic protein, proteolipid protein, majoroligodendrocytic protein, myelin associated glycoprotein, andαB-crystallin (multiple sclerosis and EAE); collagen type II, heat shockproteins, aggrecans, proteoglycans, fillagrin and link (collagen-inducedarthritis, adjuvant-induced arthritis, rheumatoid arthritis); desmin(psoriasis); S-antigen (uveitis); insulin, glutamic acid decarboxylase(NOD, type I diabetes); tropomyosin (inflammatory bowel disease);epidermal cadherin (pemphigus vulgaris); Sm, RNP, histones (systemiclupus erythematosus); thryoid stimulating hormone receptor (Grave'sdisease); thyroglobulin, peroxidase (Hashimoto's thyroiditis); collagentype IV (Goodpasture's syndrome); platelet integrin a IIb: IIIa(autoimmune thrombocytopenia purpura); Rh blood group 1 antigen(autoimmune hemolytic anemia); and acetylcholine receptor (myastheniagravis). For allotransplation, allopeptides or allogeneic T cells canserve as tolerogens.

Immunomodulatory agents that induce an active immune response includevaccines that elicit an immune response that specifically ornon-specifically targets pathogenic T cells. Non-specific vaccinesinclude, for example, vaccines containing antigens present on all ormost T cells (e.g. CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD27, CD28, CD32,CD43, and T cell receptor constant regions).

T cells express on their surface a heterodimeric T cell receptor,composed of either α/β chains or γ/δ chains. There are now known to beat least 24 β chain variable region gene families, some of which havemultiple family members, and also a large number of α chain variableregion gene families. It is well established that many autoimmune andinfectious pathologies are mediated by T cells expressing a limitedrepertoire of T cell receptors, which are clonally expanded in responseto antigen or superantigen stimulation. For example, EAE has beendemonstrated to be associated with rodent Vβ8.2 (BV8S2) expressing Tcells; psoriasis with human Vβ3, Vβ13.1 and Vβ17 expressing T cells;diabetes with human Vβ6.1, Vβ6.6/6.7 and Vβ14 expressing T cells;multiple sclerosis with Vβ2, Vβ5 (e.g. Vβ5.1 and Vβ5.2), Vβ6 (e.g.Vβ6.1, Vβ6.2, Vβ6.5, Vβ6.7), Vβ7 and Vβ13 expressing T cells; andrheumatoid arthritis and certain superantigen-mediated infectiousdiseases with Vβ3, Vβ14 and Vβ17 expressing T cells. Clonally expandedpopulations of T cells are also associated with T cell proliferativepathologies, such as T cell leukemia and T cell lymphoma.

Those skilled in the art can determine the T cell receptor or receptorspresent on the relevant pathogenic T cells for the particular pathologyor particular individual using methods described, for example, in U.S.Pat. Nos. 5,612,035; 5,861,164; 6,007,815; 5,837,246; 5,985,552;5,614,192; 5,223,426; 6,113,903, and 5,776,459, and in PCT publications95/21623; 93/06135; 94/25063; 99/27957 and 95/00658. Briefly, asdescribed in these references, T cell receptor usage by the relevant Tcells (such as CD25+ activated T cells, antigen-responsive T cells, or Tcells from the site of the pathology) can be determined by thepolymerase chain reaction using a panel of V-region specific primers, orby using anti-TCR antibodies.

Administration of T cell clones expressing T cell receptors associatedwith the particular pathology, or administration of the correspondingintact dimeric T cell receptors, full-length single T cell receptorchains or portions thereof, variable region peptides or portionsthereof, or complementarity determining region peptides (e.g. the CDR1,CDR2, CDR3 or CDR4 region) or portions thereof, and analogs of thesesequences, alone or in combination, have been shown to induce immuneresponses specific for the pathogenic T cells that reduce the activityor number of pathogenic T cells. Thus, such molecules can be used asimmunomodulatory agents in combination with low dose estrogenadministration to reduce the severity of immune pathologies.

The sequences of T cell receptor α, β, γ or δ variable and constantregions from a variety of species are well known in the art (see, forexample, Genevee et al., Eur J Immunol. 22:1261-1269 (1992); Arden etal., Immunogenetics 42:455-500 (1995); Choi et al., Proc. Natl. Acad.Sci. USA 86:8941-8945 (1989); Concannon et al., Proc. Natl. Acad. Sci.USA 83:6598-6602 (1986); Kimura et al., Eur J Immunol. 17:375-383.(1987); Robinson, J. Immunol. 146: 4392-4397 (1991); and the EMBLalignment database under alignment accession number DS23485)). Methodsof preparing and administering T cell receptors, single chains, andcharacteristic peptides therefrom, so as to stimulate an immune responsespecific for the corresponding pathogenic T cell, are described, forexample, in Example II, below, as well as in the patents and PCTpublications referenced above. Single chain peptides can contain, forexample, from about 8 to about 100 amino acids, such as from about 10 toabout 50 amino acids, including from about 15 to about 30 amino acids.

Advantageously, T cell receptor heterodimers, individual T cell receptorchains, T cell receptor variable regions, and fragments from any ofthese molecules can contain or consist of amino acid sequences from theCDR2 hypervariable region. The location of the CDR2 region for eachvariable chain is known in the art, and is generally at about amino acidresidues 38-58 of most human Vβs and Vαs. Exemplary TCR peptides thatinclude some or all of a Vβ (BV) or Vα (VA) CDR2 region are shown inTable 2:

TABLE 2 TCR Amino Acid Sequence SEQ ID NO: Vβ3 LGLRLIYFSYDVKMKEKGDI 1Vβ5.2 ALGQGPQFIFQYYEEEERQRG 2 Vβ5.2 ALGQGPQFIFQTYEEEERQRG 3 (Y49T) Vβ6.1LGQGPEFLIYFQGTGAADDSG 4 Vβ6.5 LGQGPEFLTYFQNEAQLEKS 5 Vβ13GLRLIHYSVGAGITDQGEV 6 Vβ14 SMNVEVTDKGDVPEGYK 7 Vβ17 SQIVNDFQKGDIAEGYS 8BV1S1A1N1 SLDQGLQFLIQYYNGEERAKG 9 BV1S1A2 SLDQGLQFLIHYYNGEERAKG 10BV2S1A1 FPKQSLMLMATSNEGSKATYE 11 BV2S1A3N1 FPKKSLMLMATSNEGSKATYE 12BV2S1A4T FPKQSLMLMATSNEGCKATYE 13 BV2S1A5T FPKKSLMQIATSNEGSKATYE 14BV3S1 DPGLGLRLIYFSYDVKMKEKG 15 BV4S1A1T QPGQSLTLIATANQGSEATYE 16BV5S1A1T TPGQGLQFLFEYFSETQRNKG 17 BV5S1A2T TLGQGLQFLFEYFSETQRNKG 18BV5S2 ALGQGPQFIFQYYEEEERQRG 19 BV5S3A1T VLGQGPQFIFQYYEKEERGRG 20BV5S4A1T ALGLGLQLLLWYDEGEERNRG 21 BV5S4A2T ALGLGLQFLLWYDEGEERNRG 22BV5S6A1T ALGQGPQFIFQYYREEENGRG 23 BV6S1A1N1 SLGQGPEFLIYFQGTGAADDS 24BV6S1A3T SLGQGPELLIYFQGTGAADDS 25 BV6S2A1N1T ALGQGPEFLTYFQNEAQLDKS 26BV6S3A1N1 ALGQGPEFLTYFNYEAQQDKS 27 BV6S4A1 TLGQGPEFLTYFQNEAQLEKS 28BV6S4A4T NPGQGPEFLTYFQNEAQLEKS 29 BV6S5A1N1 SLGQGLEFLIYFQGNSAPDKS 30BV6S6A1T ALGQGPEFLTYFNYEAQPDKS 31 BV6S8A2T TLGQGSEVLTYSQSDAQRDKS 32BV7S1A1N1T KAKKPPELMFVYSYEKLSINE 33 BV7S2A1N1T SAKKPLELMFVYSLEERVENN 34BV7S3A1T SAKKPLELMFVYNFKEQTENN 35 BV8S1 TMMRGLELLIYFNNNVPIDDS 36 BV8S3TMMQGLELLAYFRNRAPLDDS 37 BV9S1A1T DSKKFLKIMFSYNNKELIINE 38 BV10S1PKLEEELKFLVYFQNEELIQKA 39 BV10S2O TLEEELKFFIYFQNEEIIQKA 40 BV11S1A1TDPGMELHLIHYSYGVNSTEKG 41 BV12S1A1N1 DPGHGLRLIHYSYGVKDTDKG 42 BV12S2A1TDLGHGLRLIHYSYGVQDTNKG 43 BV12S2A2T DLGHGLRLIHYSYGVKDTNKG 44 BV12S2A3TDLGHGLRLIHYSYGVHDTNKG 45 BV12S3 DLGHGLRLIYYSAAADITDKG 46 BV13S1DPGMGLRLIHYSVGAGITDQG 47 BV13S2A1T DPGMGLRLIHYSVGEGTTAKG 48 BV13S3DPGMGLRLIYYSASEGTTDKG 49 BV13S4 DPGMGLRRIHYSVAAGITDKG 50 BV13S5DLGLGLRLIHYSNTAGTTGKG 51 BV13S6A1N1T DPGMGLKLIYYSVGAGITDKG 52 BV13S7DPGMGLRLIYYSAAAGTTDKE 53 BV14S1 DPGLGLRQIYYSMNVEVTDKG 54 BV15S1DPGLGLRLIYYSFDVKDINKG 55 BV16S1A1N1 VMGKEIKFLLHFVKESKQDES 56 BV17S1A1TDPGQGLRLIYYSQIVNDFQKG 57 BV17S1A2T DPGQGLRLIYYSHIVNDFQKG 58 BV18S1LPEEGLKFMVYLQKENIIDES 59 BV19S1P NQNKEFMLLISFQNEQVLQET 60 BV19S2ONQNKEFMFLISFQNEQVLQEM 61 BV20S1A1N1 AAGRGLQLLFYSVGIGQISSE 62 BV20S1A1N3TAAGRGLQLLFYSIGIDQISSE 63 BV21S1 ILGQGPELLVQFQDESVVDDS 64 BV21S2A1N2TNLGQGPELLIRYENEEAVDDS 65 BV21S3A1T ILGQGPKLLIQFQNNGVVDDS 66 BV22S1A1TILGQKVEFLVSFYNNEISEKS 67 BV23S1A1T GPGQDPQFFISFYEKMQSDKG 68 BV23S1A2TGPGQDPQFLISFYEKMQSDKG 69 BV24S1A1T KSSQAPKLLFHYYNKDFNNEA 70 BV24S1A2TKSSQAPKLLFHYYDKDFNNEA 71 BV25S1A1T VLKNEFKFLISFQNENVFDET 72 BV25S1A3TVLKNEFKFLVSFQNENVFDET 73 AV1S1 YPGQHLQLLLKYFSGDPLVKG 77 AV1S2A1N1TYPNQGLQLLLKYTSAATLVKG 78 AV1S2A4T YPNQGLQLLLKYTTGATLVKG 79 AV1S2A5TYPNQGLQLLLKYTSAATLVKG 80 AV1S3A1T YPNQGLQLLLKYLSGSTLVES 81 AV1S3A2TYPNQGLQLLLKYLSGSTLVKG 82 AV1S4A1N1T SPGQGLQLLLKYFSGDTLVQG 83 AV1S5HPNKGLQLLLKYTSAATLVKG 84 AV2S1A1 YSGKSPELIMFIYSNGDKEDG 85 AV2S1A2YSGKSPELIMSIYSNGDKEDG 86 AV2S2A1T YSRKGPELLMYTYSSGNKEDG 87 AV2S2A2TYSRIGPELLMYTYSSGNKEDG 88 AV2S3A1T DCRKEPKLLMSVYSSGNEDGR 89 AV3S1NSGRGLVHLILIRSNEREKHS 90 AV4S1 LPSQGPEYVIHGLTSNVNNRM 91 AV4S2A1TIHSQGPQYIIHGLKNNETNEM 92 AV4S2A3T IHSQGPQNIIHGLKNNETNEM 93 AV5S1DPGRGPVFLLLIRENEKEKRK 94 ADV6S1A1N1 SSGEMIFLIYQGSYDQQNATE 95 AV6S1A2N1SSGEMIFLIYQGSYDEQNATE 96 AV7S1A1 HDGGAPTFLSYNALDGLEETG 97 AV7S1A2HDGGAPTFLSYNGLDGLEETG 98 AV7S2 HAGEAPTFLSYNVLDGLEEKG 99 AV8S1A1ELGKRPQLIIDIRSNVGEKKD 100 AV8S1A2 ELGKGPQLIIDIRSNVGEKKD 101 AV8S2A1N1TESGKGPQFIIDIRSNMDKRQG 102 AV9S1 YSRQRLQLLLRHISRESIKGF 103 AV10S1A1EPGEGPVLLVTVVTGGEVKKL 104 AV11S1A1T FPGCAPRLLVKGSKPSQQGRY 105 AV12S1PPSGELVFLIRRNSFDEQNEI 106 AV13S1 NPWGQLINLFYIPSGTKQNGR 107 ADV14S1PPSRQMILVIRQEAYKQQNAT 108 AV15S1 EPGAGLQLLTYIFSNMDMKQD 109 AV16S1A1TYPNRGLQFLLKYITGDNLVKG 110 ADV17S1A1T FPGKGPALLIAIRPDVSEKKE 111 AV18S1ETAKTPEALFVMTLNGDEKKK 112 AV19S1 HPGGGIVSLFMLSSGKKKHGR 113 AV20S1FPSQGPRFIIQGYKTKVTNEV 114 AV21S1A1N1 YPAEGPTFLISISSIKDKNED 115AV22S1A1N1T YPGEGLQLLLKATKADDKGSN 116 AV23S1 DPGKGLTSLLLIQSSQREQTS 117AV24S1 DTGRGPVSLTIMTFSENTKSN 118 AV25S1 DPGEGPVLLIALYKAGELTSN 119 AV26S1KYGEGLIFLMMLQKGGEEKSH 120 AV27S1 DPGKSLESLFVLLSNGAVKQE 121 AV28S1A1TQEKKAPTFLFMLTSSGIEKKS 122 AV29S1A1T KHGEAPVFLMILLKGGEQMRR 123 AV29S1A2TKHGEAPVFLMILLKGGEQKGH 124 AV30S1A1T DPGKGPEFLFTLYSAGEEKEK 125 AV31S1YPSKPLQLLQRETMENSKNFG 126 AV32S1 RPGGHPVFLIQLVKSGEVKKQ 127

Exemplary combinations of TCR peptides that can be used in combinationwith low dose estrogen for preventing or reducing the severity ofautoimmune diseases include, for multiple sclerosis, Vβ5.2, Vβ6.5 and/orVβ13 (e.g. SEQ ID NOS:2, 3, 5 and/or 6); for rheumatoid arthritis, Vβ3,Vβ14 and/or Vβ17 (e.g. SEQ ID NOS:1, 7 and/or 8); and for psoriasis, Vβ3and/or Vβ13 (e.g. SEQ ID NOS:1 and/or 6). Other appropriate combinationsof TCR peptides can be determined by the skilled person according to thepathogenic TCRs expressed by the particular individual.

Alternatively, analogs of the T cell receptors and peptides can be usedin the methods of the invention As used herein, the term “T cellreceptor analog” refers to a sequence with minor modifications, so longas the analog retains the ability to induce a substantially similar cellmediated or humoral immune response against the T cell receptor as thereceptor or portion having the native sequence. A T cell receptor analogcan thus have one, two or several amino acid deletions, additions orsubstitutions, with respect to the native sequence. For example, a Tcell receptor analog can have a single amino acid substitution, orsubstitutions at 2, 3, 4 or more positions with respect to the sequenceslisted in Table 2. Such analogs can advantageously have improvedstability, bioavailability, bioactivity or immunogenicity as compared tothe native sequence. An exemplary analog of a Vβ sequence is the (Y49T)BV5S2-38-58 peptide having the amino acid sequence ALGQGPQFIFQTYEEEERQRG(SEQ ID NO:3), which is a singly substituted analog of the naturallyoccurring BV5S2-38-58 peptide.

A TCR analog can have, for example, at least 70%, such as at least 80%,90%, 95%, 98% or greater identity with the naturally occurring sequenceover the entirety of the sequence. A TCR analog can be encoded by anucleic acid sequence having at least 70%, such as at least 80%, 90%,95%, 98% or greater identity with the naturally occurring sequence overthe entirety of the sequence. Those skilled in the art can readily makeand test peptide analogs, by either in vitro or in vivo assays, todetermine whether they retain the immunological activity of thenaturally occurring sequence. Additionally, computer programs thatpredict sequences containing B and T cell epitopes are known in the art,and can be used to guide the choice of amino acid substitutions,additions or deletions (see, for example, Savoie et al., Pac. Symp.Biocomput. 1999:182-189 (1999); Cochlovius et al., J. Immunol.165:4731-4741 (2000)).

As shown in Example II, below, the combination of low dose estrogen anda Vβ8.2 peptide acted synergistically to result in complete protectionagainst the autoimmune disease EAE. Thus, T cell receptor peptidetherapies, as described above, can advantageously be combined with lowdose estrogen therapy to reduce the severity of immune pathologiesmediated by T cells expressing a limited repertoire of T cell receptors,including both autoimmune pathologies and T cell malignancies.

Alternatively, an expressible nucleic acid construct encoding an intactdimeric T cell receptor, a full-length single T cell receptor chain, avariable region peptide or portion thereof, or hypervariable regionpeptide (e.g. the CDR2 region) or portion thereof, or analog of suchsequences, can be administered to a mammal. The nucleic acid can beinserted into a plasmid vector, viral vector, or alternatively not beinserted into a vector. Those skilled in the art can determine theappropriate mammalian promoter and regulatory elements, route ofadministration and dose of nucleic acid required to induce an immuneresponse against the pathogenic T cells. Preferred routes ofadministration of an expressible nucleic acid are intramuscular andintradermal. The use of expressible nucleic acid molecules encodingpeptides to induce an immune response are described, for example, inU.S. Pat. No. 5,580,859. The use of expressible nucleic acid moleculesencoding T cell receptor peptides to elicit an immune response againstpathogenic T cells is described, for example, in U.S. Pat. Nos.5,939,400, 6,113,903 and 6,207,645.

Similar immunotherapeutic methods as described above with respect to Tcell receptors can be used to induce an immune response against an HLAmolecule associated with an immune disease. For example, expression ofHLA-DR1 and some subtypes of HLA-DR4 (eg. Dw4) are strongly associatedwith rheumatoid arthritis (RA); expression of HLA-B27 is stronglyassociated with ankylosing spondylitis and reactive arthritis;expression of HLA-DR15, DQ6 and Dw2 with multiple sclerosis (MS);HLA-DR3 and HLA-DR4 with diabetes; and HLA-DR2 and HLA-DR3 with lupus.Thus, HLA molecules associated with immune pathologies andcharacteristic portions thereof, and nucleic acid molecules encodingsuch polypeptides, can be used as immunotherapeutic agents incombination with estrogen therapy to reduce the severity of immunepathologies. The association of HLA haplotypes with immune pathologiesand methods of using HLA molecules as immunotherapeutic agents aredescribed, for example, in U.S. Pat. No. 6,045,796.

Immunomodulatory agents can advantageously be administered incombination with an adjuvant suitable for administration to theparticular mammal. For humans, exemplary adjuvants include IncompleteFreund's Adjuvant, alum, and Detox™. Optionally, immunomodulatory agentscan be conjugated to carrier molecules. Suitable adjuvants and carriersare well known in the art.

As used herein, an “immunoblocking agent” refers to any molecule thatinterferes with the interaction of the trimolecular complex between a Tcell receptor, an HLA and an antigen. For example, an immunoblockingagent can be an antibody directed against and specific for a T cellreceptor chain, such as specific for a rodent Vβ8.2, or human Vβ2, Vβ3,Vβ5.1, Vβ5.2, Vβ6.1, Vβ6.2, Vβ6.5, Vβ6.7, Vβ7, Vβ13, Vβ14 or Vβ17 chain.Methods of using antibodies as T cell receptor immunoblocking agents aredescribed, for example, in Acha-Orbea et al., Cell 54:263-273 (1988) andU.S. Pat. Nos. 5,223,426, 6,221,352 and 6,113,903.

Likewise, an immunoblocking agent can be an antibody directed against anantigen, such as the antigens associated with immune pathologiesdescribed above, or an antibody directed against an HLA antigenassociated with an immune pathology, as described above.

As used herein, the term “antibody” refers to a polyclonal, monoclonal,chimeric or single chain antibody, or antigen-binding fragment therefrom(such as as a Fab or Fab2 fragment), that binds an antigen with highaffinity (Kd<about 10⁵M) and high specificity. Methods of preparingantibodies specific for any given target molecule are well known in theart.

An immunoblocking agent can further by a complex of an antigenic peptideand an HLA molecule as described, for example, in U.S. Pat. No.5,194,425.

An immunoblocking agent can also be a non-antibody agent thatspecifically binds a desired target molecule on a pathogenic T cell (eg.T cell receptor, antigen or HLA). Libraries of naturally occurring andsynthetic compounds, including inorganic compounds, peptides, lipids,saccharides, nucleic acids and small organic molecules, are commerciallyavailable, and can be screened in high-throughput assays to bindingagents. Such agents can then be tested in in vitro or in vivo assays todetermine their efficacy in blocking activation of pathogenic T cells.

For example, an immunoblocking agent can be an altered peptide ligand.As used herein, the term “altered peptide ligand” refers to an analog ofan antigenic peptide (such as the autoantigenic peptides describedabove), in which the TCR contact residues have been altered, such thatthe peptide binds the HLA molecules with similar affinities as thewild-type peptide, but does not stimulate T cell proliferativeresponses. Methods of making and using altered peptide ligands of avariety of antigenic peptides are described, for example, in Evavold etal., Immunology Today 14:602-609 (1993), in Fairchild, Eur. J.Immunogenet. 24:155-167 (1997), and in U.S. Pat. No. 6,197,926.

Advantageously, an antibody or other immunoblocking agent can further beattached to a toxic moiety, such as a chemotherapeutic agent orradioisotope to kill or inhibit proliferation of target cells. Suchmoieties and methods of attaching them to immunoblocking agents areknown in the art.

An immunotherapeutic agent can alternatively be an agent that acts by amechanism that is not specific for the trimolecular T cellreceptor-antigen-HLA complex. Such agents include, for example, agentsthat modulate levels, production or function of cytokines, chemokines ortheir receptors. Those skilled in the art understand which sorts ofagents will be effective in relation to different immune pathologies.For example, for treatment of Th1-mediated pathologies, useful agentsinclude those that decrease Th1-type and/or increase Th2-type cytokinelevels or activity.

Binding domains from the TNFα receptor (e.g. Enbrel™), or antibodies orother agents that bind to or block the function of TNFα (e.g.etanercept; infliximab), are exemplary immunotherapeutic agents thatinhibit Th1 immune responses. Other agents include the naturallyoccurring IL-1 receptor antagonist (IL-1ra). Additionally, usefulimmunotherapeutic agents include general immunosuppressive agents suchas corticosteroids, cyclosporine and FK506; anti-inflammatory cytokinessuch as IL-4, IL-10, TGF-β and interferons (e.g. interferon (IFN)beta-1a(Avonex™); IFNbeta-1b (Betaseron™); Rebif™); agents thatnon-specifically interfere with TCR/HLA/antigen interactions (e.g. thebasic four-amino acid copolymer known as glatiramer acetate(Copaxone™)); antineoplastic agents (e.g. mitoxantrone (Novantrone™);purine analogs (e.g. 2-chlorodeoxyadenosine (cladribine);2′-deoxycorfomycin (pentostatin)) as well as methotrexate, Cox-2inhibitors (e.g. etoricoxib), phosphodiesterase inhibitors, leflunomideand the like, and various combinations of the above agents.

The invention also provides kits containing a low dose of estrogen andan immunotherapeutic agent, wherein administration of the low dose ofestrogen and the immunotherapeutic agent reduce the severity of aTh1-mediated immune pathology in a mammal. Appropriate kit componentsand immunotherapeutic agents for treatment of various pathologies havebeen described above.

As used herein, the term “kit” refers to components intended for usetogether, which may be in the same or separate containers. An indicationthat components of a kit are for use together can be, for example,packaging of containers containing the components in a single package,or labeling either or both of the components as being for use incombination, or both. Such kits can further contain written instructionsfor use of the low dose estrogen formulation and immunotherapeutic agentin combination to reduce the severity of an immune pathology. Writteninstructions can, for example, set forth the clinical indication, aswell as the amount, frequency, and method of administration of the kitcomponents.

It will be appreciated that the estrogen formulation and agentformulation need not be the same. For example, the agent can beformulated for administration by injection, or other appropriate route,whereas the estrogen can be formulated for implantation, oraladministration, inhalation or administration by another appropriateroute. Other formulations for the components of the kit can bedetermined by those skilled in the art, following the guidance providedabove in relation to methods for reducing the severity of immunepathologies.

In addition to the active ingredients, the low dose estrogen andimmunotherapeutic agent can be formulated with suitablepharmaceutically-acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Maack Publishing Co.,Easton, Pa.).

The kit components are provided in an effective amount to achieve theintended purpose. The determination of an effective dose is well withinthe capability of those skilled in the art. For any compound, thetherapeutically effective dose can be estimated initially either in cellculture assays, or in animal models, usually mice, rats, rabbits, dogs,or pigs. The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans.

A therapeutically effective dose refers to that amount of activeingredient which ameliorates the symptoms or condition. Therapeuticefficacy and toxicity can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts of the immunotherapeutic agent may vary from 0.1μg to 100 mg, up to a total dose of about 1 g, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Effect of Low-Dose Estrogen Therapy on an Immune Pathology

This example shows that administration of a low dose of either of twoforms of estrogen, to either males or females, in two distinct animalmodels of multiple sclerosis, effectively reduced the incidence andseverity of the disease. The reduction in clinical disease wasaccompanied by a significant decline in the number of inflammatory anddemyelinating foci in the central nervous system. T lymphocytes fromestrogen treated mice demonstrated a modest reduction in proliferationand a shift in cytokine production. Thus, low dose estrogen therapy isan effective method of reducing the severity of immune pathologies inmammals.

Materials and Methods

Animals. Age matched SJL/J and B10.PL mice were purchased from JacksonLaboratory (Bar Harbor, Me.). Young adult (10 weeks old or less) micewere used for the experiments in this Example. The animals were housedin the Animal Resource Facility at the Portland Veterans Affairs MedicalCenter in accordance with institutional guidelines.

Antigens. Mouse proteolipid protein peptide 139-151 (HCLGKWLGHPDKF) (SEQID NO:74) and myelin basic protein peptide Acl-11 (Ac-ASQKRPSQRSK) (SEQID NO:75) were synthesized using solid phase chemistry on a Synergy 432Apeptide synthesizer (Applied Biosystems, Foster City, Calif.), andpurified prior to use.

Estrogen treatment and measurement of serum estrogen levels. Sixty-dayrelease pellets of 17-β estradiol (E2), estriol (E3) and placebo pelletswere implanted subcutaneously in the scapular region behind the neckusing a 12 gauge trochar as described by the manufacturer (InnovativeResearch, Sarasota, Fla.). The mice were implanted one week prior toimmunization with the appropriate myelin antigen. Representative animalswere bled by cardiac puncture, and the blood was allowed to clot at 4°C. overnight. The samples were centrifuged, the sera collected, andstored at −80° C. until hormone analysis was performed. Serum levels ofE2 and E3 were determined by radioimmunoassay (RIA) after Sephadex LH-20column chromatography. All samples were analyzed in a single assay foreach hormone.

Induction of EAE. SJL mice were inoculated subcutaneously in the flankswith 0.2 ml of an emulsion containing 150 μg of PLP 139-151 in salineand an equal volume of complete Freund's Adjuvant (CFA) containing 200μg of Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit,Mich.). B10.PL mice were immunized with an emulsion containing 400 μg ofMBP Acl-11 and 200 μg of Mycobacterium tuberculosis. Disease inductionin B10.PL mice required treatment with pertussis toxin on the day ofimmunization (75 ng/mouse) and 2 days later (200 ng/mouse). The micewere examined daily for clinical signs of disease and scored accordingto the following scale: 0, normal; 1, minimal or mild hind limbweakness; 2, moderate hind limb weakness or mild ataxia; 3, moderatelysevere hind limb weakness; 4, severe hind limb weakness or moderateataxia; 5, paraplegia with no more than moderate forelimb weakness; 6,paraplegia with severe forelimb weakness or severe ataxia.

Histopathology. The intact spinal column was removed from mice duringthe peak of clinical disease and fixed in 10% phosphate bufferedformalin. The spinal cords were dissected after fixation, and embeddedin paraffin prior to sectioning. The sections were stained with luxolfast blue-periodic acid schiff-hematoxylin and analyzed by lightmicroscopy. Semi-quantitative analysis of inflammation and demyelinationwas determined by examining at least ten sections from each mouse.

Immunofluorescent staining for flow cytometry. Draining lymph node (DLN)cells were removed during the peak of clinical symptoms and analyzed forthe expression of cell surface proteins by fluorescent staining ex vivo.The following fluorochrome conjugated antibodies obtained fromPharmingen Inc. (San Diego, Calif.) were used for the direct staining ofDLN cells: anti-CD4, anti-CD25, anti-CD69, anti-CD95L, anti-CD44,anti-CD62L, anti-CD49d. Two-color immunofluorescent analysis wasperformed on a FACScan instrument (Becton Dickinson, Mountain View,Calif.) using Cellquest software. For each experiment the cells werestained with isotype control antibodies to establish backgroundstaining, and to set the quadrants prior to calculating the percentpositive staining cells.

Proliferation Assays. DLN cells were recovered from immunized mice atpeak of clinical EAE (days 12-16 post-immunization) as described in Beboet al., J. Immunol. 162:35 (1998)). The in vitro proliferative responsewas determined using a standard microtiter assay (Bourdette et al., CellImmunol. 112:351 (1988)). Briefly, DLN cells were cultured in 96 well,flat bottom tissue culture plates at 4×10⁵ cells per well in stimulationmedium alone (control), or with test antigens (i.e. PLP 139-151) andincubated for 3 days at 37° C. in 7% CO₂. Wells were pulsed for thefinal 18 hr with 0.5 mCi of [³H] methylthymidine (Amersham, ArlingtonHeights, Ill.). The cells were harvested onto glass fiber filters andtritiated thymidine uptake measured by a liquid scintillation counter.Results were determined from the means of triplicate cultures.Stimulation indices were determined by calculating the ratio of antigenspecific cpm to control cpm.

Cytokine detection by ELISA. DLN cells were cultured at 4×10⁶/ml andstimulated with the appropriate antigen in 24 well culture plates. Cellculture supernatants were recovered between 48-72 hr and frozen at −70°C. until needed for the cytokine assay. Measurement of cytokines wasperformed by ELISA using cytokine specific capture and detectionantibodies (Pharmingen). Standard curves for each assay were generatedusing recombinant mouse cytokines (Pharmingen), and the concentration ofcytokines in the cell supernatants was determined by interpolation fromthe appropriate standard curve. IFN-γ, TNF-α and IL-12 were chosen asrepresentative Th1 cytokines, while IL-4 and IL-10 were measured asrepresentative Th2 cytokines.

PLP 139-151 specific antibody ELISA. Nunc-Immuno 96 well ELISA plates(Nunc, Inc, Denmark) were coated with PLP 139-151 at 4 μg/ml inphosphate buffered saline (PBS) overnight at 4° C. The plates werewashed and blocked prior to the addition of serum at the indicateddilution in triplicates. The samples were incubated overnight at 4° C.and the plate was washed prior to the addition of an affinity purified,biotinylated goat anti-mouse Ig (diluted 1:10,000) detecting antibody(Accurate Antibodies, Westbury, N.Y.). The plates were incubated for onehour at room temperature before they were washed. A 1:400 dilution ofavidin-peroxidase conjugate (Sigma, St. Louis, Mo.) was added to eachwell and the plates were incubated for an additional 45 mins. After thefinal wash, a peroxidase substrate (3,3′, 5,5-tetramethylbenzidine,Kirkgaard & Perry Laboratories, Gaithersburg, Md.) was added to thewells and the reaction was stopped by the addition of 0.18 M sulfuricacid. The plates were read in a Vmax kinetic microplate reader(Molecular Devices, Inc., Sunnyvale, Calif.) at 450 nm. Wells coatedwith an irrelevant peptide (myelin oligodendrocyte glycoprotein 35-55)acted as a negative control.

Statistics. Cumulative disease index (CDI) was defined as the mean ofthe sum of the daily scores. Significant differences in diseaseincidence between placebo and estrogen treated mice were determined bychi square analysis and significant differences in disease onset,severity at peak of disease and CDI were determined using the two-tailedstudent t test.

Results

Low dose 17β-estradiol (E2) treatment reduces the incidence and severityof EAE in SJL mice. The protective effect of estrogen on severity ofEAE, an animal model of multiple sclerosis was determined. Female SJLmice were implanted with 60-day release tablets (Innovative Research,Sarasota, Fla.) containing 17-estradiol (E2) one week prior to theactive induction of EAE by immunization with proteolipid protein peptide139-151 (PLP 139-151). The dose of E2 chosen for these studies (Table 3)was intended to mimic the levels of E2 found during pregnancy, estrus ordiestrus phases of the hormone cycle (Table 1). E2 levels were measuredin representative animals and were determined to be equivalent to thosereported by the manufacturer.

TABLE 3 Pellet (mg) 17β-estradiol (pg/ml) Estriol (pg/ml) 15 9,000-10,000 10,000-15,000 5 3,000-4,000 5,000-6,000 2.5 1,500-2,0002,000-3,000 1.5   800-1,000   800-1,000 0.36 150-200 150-200 0.1 25-5040-50 0.025  5-10 10-20

As shown in FIG. 1 and Table 4, pregnancy levels of E2 reduced theincidence and severity of clinical disease in a manner similar to thatreported previously. Unexpectedly, low levels of E2 also profoundlyreduced the clinical manifestations of disease. Pellets releasing aslittle as 25-50 pg of E2 per ml of serum lowered the incidence, delayedthe onset, and significantly diminished the severity of paralysis whencompared to placebo controls (FIG. 1 and Table 4). In addition,pathological examination revealed a dramatic reduction in mononuclearcell infiltration and demyelination in the spinal cords of E2 protectedmice when compared to placebo treated mice (Table 5).

TABLE 4 17β-estradiol (mg/pellet) Incidence Onset (days) Relapse PeakCDI Placebo 19/19 12.3 ± 0.4 5/10 4.4 ± 0.2 30.5 ± 1.2 (100%) (50%)0.025 15/18 15.8 ± 3.5 5/9 3.6 ± 1.3 19.2 ± 10.0 (diestrus) (83%) p =0.09 (56%) p = 0.27 p = 0.07 p = 0.201^(T) p = 0.843 0.1 12/19 14.5 ±2.0 2/9 2.0 ± 0.9  8.9 ± 3.4 (diestrus) (63%) P = 0.07 (22%) P = 0.0002P < 0.0001 p = 0.012 p = 0.431 0.36 15/19 15.1 ± 1.3 2/9 2.5 ± 0.6  9.8± 1.9 (estrus) (79%) P = 0.006 (22%) P < 0.0001 P = 0.0001 p = 0.114 p =0.431 2.5  4/10 13.8 ± 0.5 0/4 2.0 ± 2.1  7.5 ± 7.9 (pregnancy) (40%) P= 0.02 (0%) p = 0.252 P = 0.06 P = 0.003 p < 0.0001

TABLE 5 Inflammatory Demyelinated foci/section foci/section Placebo 7.2± 3.4 4.4 ± 1.8 0.36 mg E2  1.3 ± 0.8* 0.5 ± 0.3  2.5 mg E2 0.6 ± 0.80.5 ± 0.4  2.5 mg E3 1.2 ± 0.6 0.8 ± 0.2  5.0 mg E3 0.9 ± 0.5 0.3 ± 0.2

Low dose estriol (E3) treatment reduces the incidence and severity ofEAE in SJL mice. Estriol (E3) is a hormone produced by the placenta, andis at its highest levels during the third trimester. The effects ofvarious doses of ES in the SJL EAE model was determined. As shown inFIG. 2 and Table 6, high dose E3 therapy effectively reduced theincidence and severity of EAE induced by active immunization of femaleSJL mice with PLP 139-151. Unexpectedly, low dose E3 treatment was alsoeffective. Treatment of female mice with 1.5 mg E3 pellets resulted inserum hormone levels that were half to a third of that known to resultfrom pregnancy (Tables 1 and 3). These mice had a lower incidence,delayed onset, and a significant reduction in the mean peak diseasescore and cumulative disease index (FIG. 2 and Table 6). The diminutionin clinical disease score was accompanied by a substantial reduction ininflammation and demyelination upon pathological examination (Table 5).The direct comparison of E2 and E3 in the same animal model also alloweddetermination of whether one form of estrogen was more or less potentthan the other form. No statistically significant differences (asdetermined by the Fisher exact test) in the incidence or severity of EAEwere found, indicating that E2 and E3 were equally protective.

TABLE 6 Estriol Onset (mg/pellet) Incidence (days) Relapse Peak CDIPlacebo 10/10 12.7 ± 0.3 2/5 4.2 ± 0.2 30.2 ± 1.5 (100%) (40%)  1.5 6/10 16.4 ± 0.7 0/3 2.0 ± 0.0  9.3 ± 2.6 (low) (60%) P = 0.02 (0%) P =0.004 P = 0.008 p = 0.094 p = 0.673  5.0  2/5 33.5 ± 5.0 0/2 1.4 ± 2.2 6.5 ± 9 (pregnancy) (40%) P = 0.03 (0%) P = 0.31 p = 0.07 p = 0.040 p =0.895 15.0  5/10 23.8 ± 9 0/1 1.4 ± 1.1  4.4 ± 3.5 (high) (50%) p = 0.22(0%) P = 0.07 P = 0.01 P = 0.039 NA

Low dose estrogen therapy reduces the incidence and severity of EAE inB10.PL mice. The effect of E2 and E3 on EAE was examined in B10.PL mice,which are genetically distinct from SJL mice and respond to a differentdominant myelin antigen, myelin basic protein peptide Acl-11 (MBPAcl-11). The sensitivity of these mice to estrogen therapy was tested bytreating the mice with estrogen containing pellets prior to immunizationwith MBP Acl-11. Low level E2 treatment reduced the incidence, delayedthe onset, and diminished the severity of EAE as reflected bysignificant differences in mean peak disease score and the cumulativedisease index (Table 7).

TABLE 7 17β- estradiol Onset (mg/pellet) Incidence (days) Peak CDTPlacebo 26/30 (87%) 13.6 ± 0.6  4.6 ± 0.34 39.3 ± 7.3  0.18  3/6 (50%)25.7 ± 1.2  1.7 ± 0.92  5.8 ± 2.9 (diestrus) p = 0.125 P < 0.0001 P <0.000 P < 0.0001  0.36 15/26 (58%) 26.5 ± 1.3 2.61 ± 0.60 13.5 ± 6.2(estrus) p = 0.030 P < 0.0001 P < 0.0001 P < 0.0001  2.50  0/15 (0%)   0± 0   0 ± 0   0 ± 0 (pregnancy) p < 0.0001 NA NA NA 15.0  2/8 (25%)   29± 1.4  2.0 ± 1.0  1.4 ± 3.8 (high) p = 0.002 P < 0.0001 P < 0.0001 P <0.0001

When the cumulative disease indices and peak disease scores werecompared (Fisher exact test), no significant differences in E2sensitivity between SJL and B10.PL mice at low E2 levels were found .However B10.PL mice appeared to be more sensitive to high dose E2treatment. Strain differences in peak disease score and cumulativedisease index were significant in mice receiving 2.5 mg E2 pellets(p=0.005).

E3 also reduced the incidence and severity of disease in B10.PL mice(Table 8), but no differences in sensitivity to E3 were detected betweenthese mice and SJL mice as determined by the Fisher exact test.

TABLE 8 Estriol Onset (mg/pellet) Incidence (days) Peak CDI Placebo 3/4(75%)  9.7 ± 0.43  4.3 ± 1.4 30.5 ± 30.5 1.5 5/8 (38%) 30.3 ± 0.9 1.38 ±0.8  1.9 ± 1.7 (low) p = 0.551^(T) P < 0.0001 P < 0.0001 P < 0.0001 53/8 (38%) 31.3 ± 0.7 0.71 ± 0.4  1.0 ± 0.5 (pregnancy) p = 0.551 P <0.0001 P < 0.0001 P < 0.0001

Male SJL mice are sensitive to estrogen. Estrogen receptors (ER) areexpressed both by female and male immunocompetent cells. Because malecells are potentially sensitive to estrogens, estrogen therapy wasperformed on male SJL mice. Male mice were treated with E2 and E3containing pellets as described above and one week later they wereimmunized with PLP 139-151. Treatment with either E2 or E3 delayed theonset and reduced the severity of clinical disease, even at dosesequivalent to estrus levels (150-200 pg/ml) in females (FIG. 3 and Table9). No significant differences in estrogen sensitivity (as determined bythe Fisher exact test) were detected between males and females.

TABLE 9 Treatment Onset (mg/pellet) Incidence (days) Peak CDI Placebo4/4 (100%) 11.3 ± 0.5  4.4 ± 0.8 25.4 ± 6.5 0.36 E2 3/4 (75%) 15.3 ± 3.2 2.1 ± 2.4  6.4 ± 5.1 p = 1.00^(T) P = 0.052 P = 0.119 P = 0.004  2.5 E20/4 (0%) NA NA NA p = 0.034  2.5 E3 2/3 (67%) 16.5 ± 0.7 0.67 ± 0.6  3.2± 2.5 p = 0.885 P < 0.0001 P = < 0.0001 P = 0.003  5.0 E3 1/4 (25%) 16.0± 0 0.50 ± 1.0  2.6 ± 5.3 p = 0.144 P < 0.0001 P = < 0.0001 P = 0.002

Mechanisms governing estrogen mediated regulation of EAE. Alterations inthe expression of adhesion or activation markers on T cells are oftenindicative of functional changes in the cell. Monoclonal antibodiesspecific for a number of these markers were used to assess whetherestrogen therapy altered their expression. Draining lymph node (DLN)cells were recovered from mice during the peak of clinical EAE,incubated with the indicated fluorochrome conjugated monoclonalantibodies, and surface expression measured by fluorescent activatedcell analyzer. As shown in Table 10, there were no apparent differencesin the number of CD4+ T cells in the DLN from placebo and estrogentreated mice.

TABLE 10 % of Total % of CD4 + T cells CD4 CD25 CD69 FASL CD44 CD62L*CD49d Placebo  42* 5.0 7.2 2.8 38 7.2 36 0.36 E2 49 5.9 6.6 2.7 43 7.045  2.5 E2 49 6.3 6.5 4.2 41 6.7 43  2.5 E3 45 5.5 7.5 3.1 36 12 36  5.0E3 47 6.4 8.3 2.7 38 8.8 37 *Representative of two differentexperiments.

Approximately 5% of the T cells in the DLN presented with an activatedphenotype (CD25+, CD69+, FASL+), but no differences between placebo andestrogen treated mice was noted. In addition, no differences in adhesionmolecule expression (CD44, CD62L, CD49d) were observed. These dataindicate that estrogen therapy had no apparent effect on the phenotypeof T lymphocytes in the lymph nodes draining the site of immunization.

Proliferation of draining lymph node (DLN) T cells from placebo andestrogen treated mice was measured to determine if estrogen therapyadhered the ability of these cells to respond to antigen. DLN T cellswere removed from representative animals during the peak of clinicalEAE, stimulated with antigen in vitro, and proliferation measured usinga standard ³H-thymidine incorporation assay. A modest decrease inproliferation to PLP 139-151 was consistently observed in DLN cellsisolated from estrogen treated mice (FIG. 4). However, in all cases thereduction in antigen specific proliferation failed to achievestatistical significance. No consistent differences in background, ormitogen-induced proliferation were observed. A similar modest butinsignificant reduction in antigen specific proliferation was alsoobserved in the B10.PL model.

To determine if low dose estrogen therapy altered cytokine secretionpatterns, DLN cells were prepared from individual mice at the peak ofclinical EAE and cytokine levels were measured 48-72 hours after invitro stimulation with PLP 139-151. IFN-γ, IL-12, and TNF-α were used asrepresentative Th1 cytokines while IL-4, and IL-10 were used asrepresentative Th2 cytokines. Even though the secretion of IFN-γ wasconsistently lower in E2 and E3 treated groups of mice (FIG. 5A), thereduction in IFN-levels fell short of being statistically significant(p>0.10). The decrease in IFN-secretion was accompanied by a modestincrease in IL-10 (FIG. 5B) and a small decrease in IL-12 (FIG. 5E).Despite the lack of statistical significance, the trend towards higherTh1 and lower Th2 cytokines points towards a subtle shift in the Th1/Th2balance. The shift can be seen more clearly when the cytokine responseof each individual mouse is plotted as a ratio of IFN-γ to IL-10 (FIG.5C). There was a marked decrease in the frequency of high Th1 respondermice in the E2 and E3 treated groups when compared to placebo animalsthat approached significance (p=0.09 for the 5.0 mg E3 treated mice).Additionally, no informative trends were detected in IL-4 secretion(FIG. 5D), and TNF-α secretion was very often below the limits ofdetection for the assay (<31.25 pg/ml). Modest changes in cytokineresponses induced by low dose estrogen therapy were also observed in theB10.PL model and were consistent with the data described for the SJLmodel.

The humoral immune response in low dose estrogen treated animals wascompared to placebo controls. Serum was collected from individual miceat the peak of clinical disease and PLP 139-151 specific immunoglobulinlevels were measured using a standard ELISA assay. No significantdifference (p>/=0.180) in PLP 139-151 specific antibody production wasobserved between placebo and estrogen treated groups (FIG. 6). Thesedata suggest that the modest shift towards Th2 cytokine production inestrogen-treated mice was insufficient to enhance humoral immunity.

EXAMPLE II Effect of the Combination of Low-Dose Estrogen andImmunotherapy on an Immune Pathology

This example shows that the combination of vaccination with a BV8S2(Vβ8.2) peptide and low-dose estrogen therapy resulted in fullprotection against disease in an animal model of multiple sclerosis,whereas only partial protection was observed with either therapy alone.Additionally, the combined effects of immunotherapy and low-doseestrogen therapy potentiated IL-10 production by regulatory T cells, andsynergistically enhanced IL-10 and TGF production by antigen-specific Tcells. Thus, low dose estrogen therapy is an effective method ofenhancing the efficacy of immunotherapeutic agents in reducing theseverity of immune pathologies in humans.

Materials and Methods

Animals. Tg mice bearing the functionally rearranged BV8S2 gene specificfor MBP-NAc1-11 on the B10.PL background were provided by Dr. JoanGoverman (Seattle, Wash.). Male Tg mice were bred with B10.PL females,and the offspring tested for expression of the transgene by FACSanalysis of blood cells stained for BV8S2 (Vβ8.2) as described inGoverman et al., Cell 72:551-560 (1993). Approximately half of eachlitter expressed the BV8S2 transgene, with approximately half of thesetransgenic littermates of each sex. For some experiments, miceexpressing the BV8S2 transgene were compared to litter mates that didnot express the transgene. The colony was housed and cared for at theAnimal Resource Facility (Portland VAMC) according to institutionalguidelines.

Antigens. N-acetylated MBP-1-11 peptide (Ac-ASQKRPSQRSK) (SEQ ID NO:75)was synthesized using solid phase techniques and was purified by highperformance liquid chromatography (HPLC) at the Beckman Institute,Stanford University (Stanford, Calif.). Glutathione S-transferase (GST)and GST-BV8S2 proteins were expressed and purified as described inVaniene et al., J. Neurosci. Res. 45:475-486 (1996). The GST-BV8S2fusion protein contains the complete BV, BD, and BJ regions and thefirst 19 residues of the BC region from the TCR of an encephalitogenicrat T cell clone fused to the C-terminal end of GST. To control for theGST-BV8S2 protein, the GST protein was produced and purified using thesame expression system. The GST protein was included as a control in alltissue culture experiments utilizing the GST-BV8S2 protein.

Induction of active EAE and protection with BV8S2 protein. EAE wasinduced in Tg male or female mice by injecting 400 g MBP-Ac1-11/CFAcontaining 200 g Mycobacterium tuberculosis s.c. over four sites on theflank. For TCR protection experiments, mice were injected with 12.5 μgrecombinant rat BV8S2 protein/IFA (experimental) or saline/IFA orGST/IFA (sham controls) intraperitoneally (i.p.) on days −7 and +3relative to injection of the MBP-NAc1-11, according to the protocoldescribed in Kumar et al., J. Exp. Med. 178:909-916 (1993). In analternative protocol, mice were given the initial two injections andthen boosted weekly with 12.5 μg BV8S2 protein or saline given s.c.Groups of male and female mice that were treated with TCR protein (FIG.7) were litter mates.

Estrogen therapy. For estrogen hormone therapy or combined estrogen plusTCR therapy, 3 mm pellets containing varying amounts of 17β-estradiol orestriol (Innovative Research of America, Sarasota, Fla.) were implantedsubcutaneously on the animal's back seven days prior to induction ofEAE. Control mice were sham operated but received no pellet. Theestrogen pellets provide continuous controlled release of a constantlevel of hormone over a period of 60 days. The concentration of17β-estradiol in pellets used in these experiments and the expectedserum concentration of secreted hormone maintained in the mice arelisted in Table 11, along with the established range of physiologicalserum hormone levels during the estrus cycle and pregnancy. Serumconcentrations of estrogen monitored prior to and during the course ofEAE in representative control and implanted mice consistently fellwithin the expected ranges.

TABLE 11 Pellet (mg) 17β-estradiol (pg/ml) Physiological Equivalent 15 9,000-10,000 Pregnancy (5,000-10,000 pg/ml) 5 3,000-4,000 2.51,500-2,000 1.5   800-1,000 0.36 150-200 Estrus (100-200 pg/ml) 0.1025-50 Diestrus (20-30 pg/ml) 0.025  5-10

Disease assessment. Mice were assessed daily for clinical signs of EAEaccording to the 7-point scale described in Example I. The cumulativedisease index (CDI) was determined for each mouse by summing the dailyclinical scores, and the mean CDI±SEM was calculated for the control andexperimental groups. The mean clinical score (MCS) was calculated foreach mouse by dividing the CDI by the duration (days) of disease, andthe mean ±SEM calculated for the control and experimental groups.

Proliferation assay. Spleens (SPL) were removed surgically, and singlecell suspensions were prepared. Proliferative responses of T cells weredetermined in 96-well microtiter plates by incubating 4×10⁵ spleen cellsplus antigen at an optimal concentration of 20 μg/well. Cultures wereincubated for 72 hr at 37° C. and 7% CO₂, the last 18 hr in the presenceof 0.5 μCi ³H-thymidine. Cells were harvested onto glass fiber filters,and thymidine uptake was determined by liquid scintillation. Meancpm±SEM were calculated from triplicate wells. The stimulation index(SI) was obtained by dividing cpm from antigen-stimulated wells by cpmfrom wells with no antigen. SI in cultures stimulated with GST alone wassubtracted from the SI induced with GST-BV8S2 protein.

Measurement of cytokine secretion. Spleen cells were suspended at 4×10⁶cells/ml in stimulation medium with and without specific antigens. Cellculture supernatants were recovered at 72 hr and frozen at −70° C. untilneeded for the cytokine assay. Measurement of cytokines was performed byELISA (Bebo et al., supra (1998)) using cytokine specific capture anddetection antibodies (PharMingen, San Diego, Calif.). Capture antibodiesfor IFN-γ, IL-10, and TGF-β were diluted to 2 μg/ml in bicarbonatecoating buffer (0.1M NaHCO₃, pH 8.2). Standard curves for each assaywere generated using recombinant mouse cytokines (PharMingen), and theconcentration of cytokines in the cell supernatants was determined byinterpolation from the appropriate standard curve.

Assessment of antibody responses. Antibody reactivity to MBP-Ac1-11peptide and GST-BV8S2 protein was determined by indirect ELISA asdescribed in Hashim et al., J. Immunol. 144:4621-4627 (1990). Briefly,mouse antisera from treated and control Tg mice with EAE were incubatedin antigen coated wells, and bound antibody was detectedspectrophotometrically with peroxidase-labeled rabbit anti-mouseantibody and o-phenylene-diamine as a substrate. Differences betweengroups were determined using Student's t-test.

Ovariectomy. The ovaries were removed by making two bilateral incisions(5 mm) halfway between the base of the tail and the middle of the back,followed by small incisions (2.5 mm) through the peritoneal wall. Theovaries were pulled through the incisions by grasping the periovarianfat, the blood vessels ligated, and the ovaries removed. The incisionwas closed by surgical skin clips. The animals were allowed to recoverfor at least 1 week before initiation of experiments.

Androgen and estrogen detection. Mice were bled by cardiac puncture andthe blood was allowed to clot at 4° C. overnight. The samples werecentrifuged, and the sera collected and stored at −80° C. until hormoneanalysis was performed. Serum levels of estrogen were determined byradioimmunoassay (RIA) after Sephadex LH-20 column chromatography, asdescribed in Roselli et al., Endocrine 64:139 (1996). All samples wereanalyzed in a single assay.

Results

Gender difference in treatment of EAE with BV8S2 protein. Responses tovaccination with BV8S2 protein were compared in male versus female Tgmouse littermates, using two different protocols. As is shown in FIG. 7Aand quantified in Table 12, males injected i.p. with BV8S2 protein/IFAon days −7 and +3 relative to EAE induction were significantly protectedfrom EAE, with lower incidence and cumulative disease scores (CDI) thansham treated males. In contrast, females vaccinated using the sameprotocol were not protected from EAE (FIG. 7C and Table 12). Asillustrated in FIGS. 7B and 7E, the protective effect in males could beenhanced by boosting weekly with additional s.c. injections of BV8S2protein.

These boosting injections had an early effect on littermate females aswell, producing a significant delay in onset of clinical disease (FIG.7D and Table 12). However, this temporary suppression of clinicaldisease was lost abruptly (FIG. 7D), and there was no significantamelioration of subsequent disease assessed during days 18-30 (Table12).

TABLE 12 Treatment Inci- Day of Group Figure Group dence Onset CDI Males1A Controls 13/16 15 ± 1 49 ± 10 BV8S2  6/16* 14 ± 1 17 ± 7* Males 1BControls 10/10 13 ± 1 63 ± 9 BV8S2, B  1/9*** 15  7 ± 7*** Females 1CControls  5/5 15 ± 4 75 ± 12 BV8S2  6/6 13 ± 1 82 ± 23 Day 1-21 Day22-30 Females 1D Controls  6/6 11 ± 1 43 ± 5 35 ± 5 GST  6/6 12 ± 1 36 ±4 34 ± 3 BV8S2, B  7/7 19 ± 5** 10 ± 7** 37 ± 3

Effects of estrogen on EAE. The effects of sex hormones, including17β-estradiol and estriol, were evaluated on the clinical course of EAEby hormone depletion or addition experiments. As shown in FIG. 8A,female Tg mice unable to produce detectable levels of estrogen (<1pg/ml) or other sex hormones after ovariectomy developed significantlymore severe EAE than sham ovariectomized females (CDI=81 versus 56,p<0.001). These data demonstrate that even basal levels of ovarianfactors, possibly including estrogen, provide some regulation of EAE.

Treatment of sham or non-ovariectomized females with 17β-estradiolpellets produced a dose-dependent inhibition of EAE in both Tg females(FIG. 8A) and B10.PL littermate females (FIG. 8B). Notably, addedestrogen had a less pronounced effect on the Tg versus non-Tg females.In B10.PL females, essentially complete inhibition of EAE was producedwith 15 mg pellets secreting pregnancy levels of 17β-estradiol(9,000-10,000 pg/ml serum) over a 60 day period or with 2.5 mg pellets(1,500-2,000 pg/ml serum), and substantial inhibition was produced overa wide range of estrogen concentrations from estrus (0.36 mg pelletssecreting 150-200 pg/ml serum) to diestrus levels (0.10 mg pelletssecreting 25-50 pg/ml serum, FIG. 8B). By comparison, in Tg females,pregnancy levels of hormone (15 mg pellets) produced a marked butincomplete inhibition of EAE, with estrus levels (0.36 mg pellets)producing only moderate inhibition (FIG. 8A).

Estriol, which is normally elevated only during pregnancy, had anequivalent inhibitory effect on EAE in B10.PL females as 17β-estradiol(FIG. 9B). The partial resistance to estrogen therapy in Tg females maybe due to the higher native frequency of Ac1-11 specific T cellsafforded by the transgene (Offner et al. supra (1998)) that likelyaccounts for an increased severity of EAE (CDI=56 in Tg vs 39 in non-Tgfemales, p<0.001). Consistent with this notion, estrogen treatment ofovariectomized female Tg mice reduced the severity of EAE to about thesame level as sham ovariectomized Tg mice treated with estrogen (FIG.8A). However, the inability of estrogen to fully inhibit EAE in Tgfemales at very high levels approximating pregnancy suggests that aportion of the encephalitogenic cascade is estrogen insensitive.

Combined TCR and estrogen therapy. Because TCR and estrogen therapy wereboth partially effective for preventing EAE in Tg females, the effectsof single versus combined therapy were directly compared. Consistentwith the results described above, Tg females vaccinated with BV8S2protein (with weekly boosting) had delayed onset but eventuallydeveloped severe EAE, whereas mice treated with estrus levels of E2(0.36 mg pellets secreting 150-200 pg/ml serum over 60 days) had normalonset but generally less severe disease (FIG. 9). However, combinedtreatment with both TCR protein and estrus levels of E2 produced almostcomplete protection against EAE (FIG. 9), with only 3 of 16 micedeveloping very mild disease (Table 13). A similar degree of protectionwas provided in ovariectomized Tg females treated with BV8S2 protein andestrogen (Table 13), demonstrating that the enhanced therapeutic effectwas dependent on E2 rather than a combination of estrogen and othergonadally produced sex hormones.

TABLE 13 Total Incidence Onset Peak Mortality Average CDI (10-30)Control 60/64 11.8 ± 1.1 4.7 ± 0.1 12/49 56.4 ± 7.0 BV8S2 45/49 17.4 ±2.2^(A) 3.9 ± 1.1^(A)  1/49 32.0 ± 13.2^(A) 17β-estradiol 25/33 15.1 ±2.1A 3.6 ± 0.7^(A)  1/33 37.3 ± 6.2^(A) BV8S2-17β-  3/16 18.8 ± 8.0^(A)0.5 ± 0.4^(A)  1/16  3.1 ± 4.0^(A) estradiol Ovx 27/27 10.6 ± 0.9 5.3 ±0.7  7/27 81.2 ± 17.3^(A,C) BV8S2 23/26 18.0 ± 1.9 3.9 ± 1.3  4/26 40.1± 20.9^(B) 17β-estradiol 11/15 13.6 ± 1.5 3.5 ± 0.8  4/15 40.0 ±22.1^(B) BV8S2 + 17β-  3/16 20.8 ± 6.0 0.9 ± 0.9  0/16  7.4 ± 6.7^(A,B)estradiol Data are combined from a total of 10 separate experiments.^(A)Significant difference between control and experimental (P <0.0001). ^(B)Significant difference between Ovx control and Ovxexperimental (P < 0.0001). ^(C)Significant diifference between Ovx andnon-Ovx control (P < 0.001). Ovx, ovarectomized.

Estrogen skews response to Ac1-11 and potentiates response to BV8S2protein. To investigate the mechanism(s) involved with individual andcombined therapies, proliferation and cytokine responses of immune Tcells from naive and treated mice were evaluated. As shown in FIG. 10A,BV8S2 naive Tg males and females had equivalent proliferation responsesto Ac1-11 peptide, PPD, and ConA, but naive Tg females had a strikinglyreduced reactivity to the BV8S2 protein. This finding suggests that Tgfemales have a diminished native capacity to regulate anencephalitogenic response.

During development of EAE, splenic T cell responses to Ac1-11 peptidewere characterized by moderate proliferation and production of TGF-β,and essentially absent secretion of IL-10 (FIG. 10B). Secretion of IFN-γin response to Ac1-11 peptide was modest, reflecting preferentialmigration of inflammatory T cells to draining lymph nodes and the CNS asobserved previously(Offner et al. supra (1998)). Treatment with eitherBV8S2 protein or estrogen alone reduced proliferation and marginallyaffected cytokine responses to Ac1-11. However, combined treatment withboth BV8S2 protein and estrogen markedly reduced proliferation anddramatically enhanced production of IL-10 and TGF-β, but not IFN-γ inresponse to Ac1-11 peptide (FIG. 10B).

In contrast, splenic proliferation and IL-10 responses to BV8S2 proteinwere enhanced by both treatments individually, and further potentiatedwith combination therapy, with no significant effects of treatment onIFN- and TGF-secretion (FIG. 10C).

Additionally, combination therapy reduced circulating levels of Ac1-11specific IgG2a antibody associated with Th1 response, with no effect onIgG1 response (FIG. 10D).

EXAMPLE III 17β-estradiol Inhibits Chemokines and Receptors in EAE

This example shows that the protective effect of low dose estrogen onanimals with a Th1 immune pathology is mediated, in part, by inhibitionof mRNA expression of chemokines, chemokine receptors, and inflammatorycytokines by recruited inflammatory T cells.

Materials and Methods

Mice. Transgenic mice were obtained, bred and housed essentially asdescribed in Example II. Mice were used at 8-12 weeks of age.

Induction of active EAE. EAE was induced, and disease assessed,essentially as described in Example II. Disease onset was defined as thefirst day of clinical signs, peak—acute phase of EAE—as maximum severityof clinical signs (day 16-17 after immunization with encephalitogenicpeptide) and recovery as day 28 post-immunization, when clinicalseverity of EAE was diminished. The cumulative disease index (CDI) wasdetermined for each mouse by summing the daily clinical scores. Miceselected from control, ovariectomized, and 17β-estradiol treated groupswere sacrificed and spinal cords were isolated by insufflation andfrozen at −70C. or mononuclear cells were isolated over a Percoll stepgradient and counted as described in Offner et al., J. Immunol.161:2178-2186 (1998). Lymph nodes (LN) or spleens (SPL) were removedsurgically and passed through a wire mesh screen to obtain a single-cellsuspension. Frozen spinal cords were subsequently thawed and evaluatedfor expression of chemokines, chemokine receptors and cytokines by theRNase protection assay.

17β-estradiol treatment. For 17β-estradiol hormone therapy, 3-mm pelletscontaining 0.36 mg of 17β-estradiol (Innovative Research of America,Sarasota, Fla.), expected to provide physiological equivalence of theestrus cycle (150-200 pg/ml in serum) over 60 days, were implanted s.c.on the animal's back 7 days before induction of EAE. Control mice weresham operated and implanted with a pellet containing saline (which didnot affect the course of EAE) or no pellet. The 17β-estradiol pelletsprovide continuous controlled release of a constant level of hormoneover a period of 60 days. Serum concentrations of 17β-estradiol wereevaluated in representative mice from each group. Sham treated mice hadvariable levels of 17β-estradiol that reflected both estrus and diestrusmice (ranging from about 20 to 200 pg/ml, on average about 50 pg/ml). Incontrast, OVX mice did not have any detectable estradiol (<1 pg/ml).E2-treated intact mice were also variable (about 200-400 pg/ml),reflecting the combination of native estrus/diestrus levels plus theexogenous estradiol from the pellet, whereas the E2-treated OVX micewere somewhat lower (about 150-200 pg/ml).

RNase protection assay. Total RNA was extracted from frozen spinal cordsor lymph node cells using the STAT-60 reagent (Tel-Test, Inc.,Friendswood, Tex.). Chemokine expression was determined by using theRiboQuant RPA kit (PharMingen) according to the manufacturer'sinstructions. A multiprobe set detected the following chemokinetranscripts: C-X-C chemokines: MIP-2 and IP-10; C-C chemokines: RANTES,MIP-1α, MCP-1, and TCA-3; and C chemokine: Ltn. The chemokine receptorset detected the following transcripts: CCR1, CCR1b, CCR2, CCR3, CCR4,CCR5. Using RPA multiprobe it was possible to detect the followingcytokines: IL-4, IL-10, TNF-α, LTβ, IFN-γ. The sample loading wasnormalized by the housekeeping gene, L32, included in each template set.RPA analysis was performed on 10 μg total RNA hybridized with probeslabeled with [³²P]UTP. After digestion of ssRNA, the RNA pellet wassolubilized and resolved on a 5% sequencing gel. Controls included theprobe set hybridized to transfer RNA only, appropriate control RNA whichserves as integrity control for the RNA sample, and yeast tRNA as abackground control. For quantification, gels were exposed byphosphorimaging (Bio-Rad Laboratories, Hercules, Calif.) andradioactivity in individual bands (after background subtraction) incomparison with L32 was assessed with Quantity One software (Bio-RadLaboratories, Hercules, Calif.).

Proliferation assay. Proliferation responses of splenic T cells weredetermined essentially as described in Example II, except that antigenwas used at an optimal concentration of 50 μg/ml.

Flow cytometry. Lymph node cells from sham and 17β-estradiol treatedanimals with EAE were washed with PBS/2% FCS/0.2% NaN₃ and firstincubated on ice for 30 min with Fcγ III/II receptor blocking monoclonalantibody (PharMingen), then stained with PE-conjugated anti-CD3(PharMingen). After 20 min of incubation with anti-CD3, the cells werefixed and permablized using Cytofix/Cytoperm (PharMingen). Subsequently,cells were incubated with anti-CCR1, anti-CCR2, anti-CCR3, anti-CCR4 andanti-CCR5 goat anti-mouse polyclonal antibodies (Santa CruzBiotechnology Inc.; USA) for 30 min on ice. After washing, cells werestained with FITC-conjugated anti-goat monoclonal antibodies (Sigma) foran additional 30 min on ice. Cells were analyzed using a FACS-Scan(Becton Dickinson). Propidium iodide and forward/side scatter gatingwere used to exclude dead cells.

Measurement of cytokine secretion. Lymph nodes of naïve BV8S2 TCRtransgenic females were suspended at 4×10⁶ cells/ml in stimulationmedium with antigen and with or without 2000 pg/ml of 17β-estradiol.Cell culture supernatants were recovered at 72 hours and frozen at −70 Cuntil used. Measurement of cytokines was performed by ELISA essentiallyas described in Example II.

Ovariectomy. Ovariectomy was performed essentially as described inExample II.

Statistics. Non-parametric clinical EAE data (peak disease scores andcumulative disease index) were evaluated between groups using theMann-Whitney test; the day of onset among the various groups wasevaluated using the t-test or ANOVA; the incidence and mortality rateswere compared using the 2 test (Fisher's exact test in some instances).Comparison of RPA values, cytokine values, and CPMs were evaluated bythe t-test or ANOVA. The accepted level of significance was p<0.05.

Results

Effects of 17β-estradiol on EAE. 17β-estradiol (E2) or estriol releasedfrom implanted pellets was shown in Example I to partially inhibit EAEin a dose dependent manner in BV8S2 transgenic mice. Clinical EAE datafor groups of these mice used in the current study are graphed in FIG.11 and summarized in Table 14. Mice implanted with 0.36 mg pellets of17β-estradiol, which in combination with native hormone (20-200 pg/ml insham mice) provided 150-400 pg/ml E2 in serum (estrus levels), developedsignificantly later onset and less severe EAE (lower peak score and CDI)than sham operated control mice (FIG. 11A and Table 14). In addition toreducing disease severity, 17β-estradiol treatment inhibitedproliferation of MBP-Ac1-11 specific T cells by an average of about 47%,and prevented infiltration of mononuclear cells into spinal cords byapproximately 60% (Table 15).

In contrast, ovariectomized mice, in which endogenous 17β-estradiol wasnot detectable (<1 pg/ml), developed significantly earlier onset andmore severe signs of EAE than sham operated mice (FIG. 11B and Table14). 17β-estradiol treatment of ovariectomized mice with implanted 0.36mg pellets provided about 100-200 pg/ml E2 in serum, and inhibited EAEto approximately the same degree as E2-treated non-ovariectomized mice(FIG. 11C and Table 14).

TABLE 14 Group of mice Incidence Onset Peak CDI Sham 19/19 11.4 ± 0.54.3 ± 0.3 64.9 ± 4.7 17β-estradiol 15/18 17.4 ± 1.8* 2.5 ± 0.5** 40.4 ±7.1** treated Sham 12/12 11.3 ± 0.7 3.8 ± 0.5 59.4 ± 4.9 OVX 12/12  9.8± 0.6* 4.8 ± 0.4* 85.3 ± 7.3** OVX 12/12  9.8 ± 0.6 4.8 ± 0.4 85.3 ± 7.3OVX + 17β-  8/11 16.5 ± 2.3** 2.6 ± 0.6** 40.0 ± 8.5** estradiol^(a) *p< 0.05 **p < 0.01 ^(a)EAE severity also significantly less than in Shamgroup

TABLE 15 Group of mice Mean clinical score* Number of cells/cord Sham2.4 27000 17β-estradiol treated 1.3 10000 *mean clinical score at timeof cord removal

17β-estradiol treatment results in downregulation of chemokine mRNAexpression in spinal cords. To optimize detection of clinically-relatedchanges, chemokine expression was compared in 17β-estradiol protectedversus sham pellet implanted mice during the peak acute phase of disease(16-17 days after immunization with MBP-Ac1-11 peptide/CFA) and duringthe chronic phase (28 days after immunization). At the earlier timepoint, spinal cord (SC) tissue was sampled from sham-treated miceexhibiting paralytic EAE (scores of 4-5) and compared to SC tissue from17β-estradiol treated mice which had not yet developed any clinicalsigns (EAE scores of 0). A quantitative method—the RNase protectionassay—was employed to examine RNA synthesis for the followingchemokines: RANTES, MIP-1α, MCP-1, and TCA-3 (C-C-subfamily); and MIP-2and IP-10 (C-X-C-subfamily). The tissue sample for RPA was prepared byhomogenization of whole spinal cord and total cellular RNA wasextracted.

As quantified in FIG. 12, transcripts for RANTES, MIP-1α, MIP-2, IP-10and MCP-1, but not TCA-3 were detected in SC from Tg females at the peakand chronic phases of disease. Paraplegic mice (sham group) had abundantRNA expression of RANTES, IP-10, and MCP-1, with lesser mRNA levels ofMIP-1α and MIP-2 (FIG. 12). 17β-estradiol protected mice had profoundlylower levels of mRNA expression of all detectable chemokines. At thepeak of disease, the difference between groups reached p<0.001 (FIG.12). Differences in chemokine expression between sham and E2-treatedgroups were still present but less pronounced during the chronic phaseof EAE, reflecting the clinical status of the donors (sham, EAE scoresof 3; E2, EAE scores of 0-1).

Ovariectomy increases mRNA expression of MIP-1α and MIP-2. Ovariectomyresulted in loss of detectable 17β-estradiol, as well as other ovarianhormones, and significantly enhanced the clinical severity of EAE (FIG.11A and Table 14), implicating basal levels of these factors in naturalregulation. To evaluate effects of hormone depletion on chemokine levelsduring EAE, an RPA analysis was carried out of SC from ovariectomizedfemale mice on day 16 after induction of EAE. SC were sampled fromsham-treated mice with EAE scores of 4-5, and from ovariectomized micewith EAE scores of 5-6). Surprisingly, ovariectomized mice thatdisplayed the most severe signs of EAE had lower mRNA levels than shamtreated mice of the normally predominant chemokines RANTES, IP-10 andMCP-1, but significantly enhanced expression of MIP-1α and MIP-2 (FIG.12). 17β-estradiol treatment of ovariectomized females (that inhibitedEAE to a comparable degree as E2-treatment of intact mice), stronglyinhibited expression of all detectable chemokines (FIG. 12), againreflective of the sampling of SC from mice that had not yet developedovert clinical disease (EAE scores of 0). These results demonstrate thecapacity of supplemental 17β-estradiol to profoundly inhibit chemokineexpression, and implicate ovarian factors, including 17β-estradiol, asnatural regulators of MIP-1α and MIP-2.

MIP-1α and MIP-2 are produced by infiltrating mononuclear cells in CNS.To discern which chemokines were produced by infiltrating cells withinthe CNS, expression of chemokines in whole CNS tissue versus isolatedCNS mononuclear cells in Sham treated mice at the peak of EAE werecompared. As quantified in FIG. 13, message for MIP-1α and MIP-2, butnot RANTES, IP-10 and MCP-1, was enriched in the CNS mononuclear cellfraction, whereas message for RANTES was reduced in the mononuclear cellfraction.

17β-estradiol therapy reduces chemokine receptor mRNA expression in CNS.In addition to chemokines, expression of chemokine receptors in spinalcords of 17β-estradiol treated and control BV8S2 transgenic mice duringEAE was assessed. As quantified in FIG. 14, message for CCR1, CCR2 andCCR5 was clearly enhanced in SC samples during both peak and chronicphases of EAE, whereas message for CCR1β, CCR3 and CCR4 was notdetectable at either time point. 17β-estradiol treatment that preventedEAE strongly inhibited the elevated mRNA levels for CCR1, CCR2 and CCR5observed in sham treated mice with EAE (p<0.001). Ovariectomized micehad reduced levels of message for CCR1 and CCR2, with a lesser effect onCCR5 compared to sham-implanted control mice, and supplemental17β-estradiol treatment again inhibited expression of all of thesechemokine receptors (FIG. 14).

Down-regulation of CCR1 and CCR5 in lymphocytes isolated from peripherallymph nodes of females treated with 17β-estradiol. The reduction ofchemokine receptors in SC of 17β-estradiol treated mice raised thepossibility that there might be a systemic effect of 17β-estradioltherapy on chemokine receptor expression by lymphocytes. Thus, chemokinereceptors on lymph node cells from 17β-estradiol versus sham treatedmice with EAE were quantified using specific antibody staining and FACSanalysis. Lymph node CD3+ T cells from 17β-estradiol treated mice hadreduced mean channel fluorescence and a significantly lower percentageof positive cells when stained with antibodies to CCR1 (77+2 vs 91+6%,p=0.02) and CCR5 (27+2 vs 35+4, p=0.03) compared to T cells from shamtreated mice. No difference was observed in CCR2 staining, and CCR3 andCCR4 were not detectable.

17β-estradiol down-regulates Th1 cytokines but does not cause Th2cytokine switch. Two possible effects of 17β-estradiol therapy are 1)direct inhibition of inflammatory cytokines or 2) enhancement of Th2cells and cytokines (Th2 switch) that could locally inhibit Th1 cells.RPA analysis of cytokine message revealed predominant expression ofLT-β, TNF-α, and IFN-γ in the SC of mice at the peak of EAE that wassignificantly inhibited in 17β-estradiol treated mice (LT-β, p<0.02;TNF-α, p<0.0001; IFN-γ, p<0.01, FIG. 15). In contrast, message for IL-4and IL-10 was not detectable in SC of mice with EAE, nor were thesecytokines induced by 17β-estradiol treatment, indicating no Th2 switchin CNS. No changes in cytokine expression were noted in lymph node cellsfrom E2-treated versus sham control groups.

17β-estradiol exerts little effect on T cells cultured in vitro. Toevaluate 17β-estradiol effects on antigen specific T cells, splenocytesfrom naïve BV8S2 single transgenic female mice were stimulated withMBP-Ac1-11 peptide or BV8S2 protein in the presence or absence of arange of 17β-estradiol concentrations. As is shown in FIG. 16A, 15-2,000pg/ml 17β-estradiol had no significant effect on native T cellproliferation response to the encephalitogenic MBP-Ac1-11 peptide, and amodest enhancing effect on the native response to the BV8S2 protein. Arelatively high dose (2,000 pg/ml) of, 17β-estradiol mediated a 50%reduction of secreted IFN-γ protein, with no effects on IL-12 or IL-10secretion, by MBP-Ac1-11 stimulated LN cells from naïve BV8S2 transgenicmice (p<0.005, FIG. 16B). However, lower doses of 17β-estradiolcomparable to serum levels in the mice studied above (150-400 pg/ml) hadno inhibitory effects on IFN-γ or other cytokine secretion in vitro.

EXAMPLE IV Low Dose Estrogen Down-Regulates TNFα Production

This example shows that the protective effect of low dose estrogen onanimals with a Th1 immune pathology is mediated, in part, bydown-regulation of TNF-α secretion at the site of the pathology.

Materials and Methods

Mice. Female C57BL/6, IL-4 KO (B6.129P2-Il4^(tmlCgn)), IL-10 KO(C57BL/6-Il10^(tmlCgn)), and IFN-γ KO (B6.129S7-Ifng^(tmlTs)) mice wereobtained from The Jackson Laboratory (Bar Harbor, Me.). The mice werehoused in the Animal Resource Facility at the Portland Veterans AffairsMedical Center in accordance with institutional guidelines.

Antigens. Mouse myelin oligodendrocyte glycoprotein (MOG) 35-55(MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO:76)) was synthesized using solid phasetechniques and purified by high performance liquid chromatography (HPLC)at the Beckman Institute, Stanford University (Palo Alto, Calif.).

Estrogen treatment. Sixty-day release pellets containing 2.5 mg of17β-estradiol (E2) or vehicle were implanted subcutaneously (s.c) in thescapular region behind the neck using a 12-gauge trochar as described bythe manufacturer (Innovative Research of America, Sarasota, Fla.). Themice were implanted one week prior to immunization with MOG 35-55. Theconcentration of E2 expected in the serum is between 1,500-2,000 pg/ml,which is approximately 5 times less than the levels found duringpregnancy. E2 levels measured previously as described in Example I werefound to be equivalent to those reported by the manufacturer.

Induction of EAE. C57BL/6 and cytokine deficient mice were inoculateds.c in the flanks with 0.2 ml of an emulsion containing 200 μg of MOG35-55 in saline and an equal volume of complete Freund's Adjuvant (CFA)containing 400 μg of Mycobacterium tuberculosis H37RA (DifcoLaboratories, Detroit, Mich.). Disease induction required i.v.administration of pertussis toxin on the day of immunization (25ng/mouse) and 2 days later (67 ng/mouse). The mice were assessed dailyfor clinical signs of EAE according to the 7 point scale described inExample I.

Histopathology. Histopathologic assessment of spinal column section wasperformed essentially as described in Example I, except that sectionswere stained with either luxol fast blue-periodic acidschiff-hematoxylin or silver nitrate prior to analysis.

RNAse protection assay. Chemokine, chemokine receptor and cytokine mRNAswere detected by RPA essentially as described in Example III, exceptthat analysis was performed on 20 μg total RNA.

Proliferation assay. Proliferation assays were performed on draininglymph node (DLN) and spleen (SP) cells essentially as described inExamples I and II, using MOG 35-55 as the test antigen.

Intracellular staining for cytokines. Single cell suspensions fromspleen were prepared from immunized mice and cultured at 10×10⁶ cells/mlin stimulation media containing 50 μg/ml of MOG 35-55. The cells werestimulated for 24 hrs, the last 5 hrs in the presence of Brefeldin A.The cells were then stained with anti-Vβ8.1/8.2 TCR FITC for 30 min at4° C. prior to fixation and permeabilization with cytofix/cytopermsolution (Pharmingen, Inc.). The cells were then stained withanti-cytokine antibodies labeled with phycoerythrin (anti-mouse IFN-γ,TNF-α, IL-4, IL-10, and IL-12 from Pharmingen Inc.) for 30 min at 4° C.The cells were washed twice in perm/wash buffer (Pharmingen, Inc.) andonce in FACS staining buffer (PBS, 1% BSA, 0.05% NaN₃) prior totwo-color FACS analysis on a FACScan instrument (Beckton-Dickenson,Inc., Sunnyvale, Calif.) using Cell Quest software (Beckton-Dickenson,Inc.). For each experiment the cells were stained with isotype controlantibodies to establish background staining and to set the quadrantsprior to calculating the percent positive cells.

CNS mononuclear cells were isolated from perfused brain and spinal cordby percol gradient centrifugation as described in Bebo et al., J.Neurosci. Res. 52:420-429 (1998). The cells were stimulated withMOG-35-55 peptide for 24 hrs, the last 5 hrs in the presence ofBrefeldin A. The cells were then stained with anti-CD4 cychrome labeledantibodies prior to fixation and permeabilization. The cells weresubsequently stained with Vβ8.1/8.2 TCR-FITC and the indicated cytokinespecific antibody coupled to PE and analyzed by three-color flowcytometry. For each experiment the cells were stained with isotypecontrol antibodies to establish background staining and to set thequadrants prior to calculating the percent positive cells.

Statistical analysis. Significant differences in incidence and mortalitybetween untreated and E2 treated mice were assessed by Chi-squareanalysis. Difference in onset was determined using the two-tailedStudent t test. Differences in peak score and cumulative disease index(CDI) were assessed by the Mann-Whitney test. Statistical significanceof the frequency of cytokine secreting cells was analyzed usingStudent's t test for comparisons of two means. Differences in theexpression of chemokine and cytokine mRNA were also determined using theStudent t test. Values of p<0.05 were considered significant.

Results

Estrogen treatment reduces the severity of EAE in C57BL/6 and cytokinedeficient mice. The role of regulatory cytokines in estrogen-inducedprotection from EAE was examined using cytokine deficient mice. Asdescribed in Examples I-III, immunization with MOG 35-55 resulted in theinduction of severe EAE in wild type (WT) C57BL/6 mice. No differencesin disease severity were found in similarly immunized cytokine deficientmice (FIG. 17 and Table 16). C57BL/6 mice implanted with 17β-estradiol(E2)-containing pellets had a lower incidence of EAE, and developeddisease much later than untreated mice. However, EAE that eventuallydeveloped in some E2-treated mice was essentially equivalent in severityto untreated animals, possibly due to early depletion of the E2 pellets.Nevertheless, treatment with E2 exerted a profound reduction in both theincidence and cumulative disease index (CDI) of EAE, and significantlydelayed onset of symptoms in those mice that eventually developeddisease. Estrogen treatment had similar effects on mice deficient inIL-4, IL-10 and IFN-γ (FIG. 17 and Table 16). No statisticallysignificant differences in the ability of E2 to protect cytokinedeficient mice were found (as determined by the Fisher exact test).

TABLE 16 Inci- Peak E2 dence Onset Mortality Score CDI B6 − 29/31 10.9 ±1.9 3/31 5.1 ± 0.9 64.7 ± 27.0 + 19/31 20.8 ± 3.7 0/31 4.2 ± 1.2 15.1 ±17.0 p = P < 0.0001 p = 0.238 P = 0.222 P < 0.0001 0.005* IL-4 − 11/1111.5 ± 2.3 3/11 5.5 ± 0.4 76.7 ± 20.1 KO +  8/11 20.6 ± 4.9 0/11 4.4 ±1.0 18.4 ± 22.2 p = P < 0.0001 p = 0.214 P = 0.561 P < 0.0001 0.214IL-10 − 15/16 12.3 ± 1.3 2/16 4.9 ± 1.1 53.0 ± 27.2 KO +  6/17 22.0 ±4.3 0/17 4.2 ± 0.8  6.5 ± 10.9 p = P < 0.0001 p = 0.227 P = 0.977 P <0.0001 0.001 IFN_(Υ) − 11/11 13.6 ± 2.6 1/11 4.9 ± 1.1 58.6 ± 22.4 KO + 8/10 21.8 ± 2.7 1/10 4.5 ± 1.2 19.3 ± 15.9 p = P < 0.0001 p = 1.000 P =0.999 P < 0.018 0.214

MOG 35-55 immunized C57BL/6 and cytokine deficient mice had numerousinflammatory and demyelinating lesions in the spinal cord at the peak ofEAE, and no significant differences in the number and size of thelesions were observed. Healthy C57BL/6 and cytokine deficient mice thatwere treated with E2 before immunization did not have any detectablelesions in the spinal cord (Table 17). Thus, it is apparent from thesedata that E2 can suppress the development of both the clinical andhistopathological manifestations of EAE in the absence of IL-4, IL-10,or IFN-γ.

TABLE 17 Treatment with E2 Inflammatory Foci/Section* C57BL/6 + 0.0 ±0.0 − 5.7 ± 1.7 IFN-ΥKO + 0.0 ± 0.0 − 5.7 ± 4.1 IL-4KO + 0.0 ± 0.0 − 7.0± 1.5 IL-10KO + 0.0 ± 0.0 − 8.0 ± 1.9 Inflammatory foci were enumeratedfrom between 7-10 sections per spinal cord, at least two spinal cordswere examined per group.

Estrogen treatment reduces chemokine and chemokine receptor mRNAexpression in the CNS. The egress of inflammatory cells into the CNS isa critical first step in the development of EAE. Chemokines are lowmolecular weight chemotactic molecules that are thought to play animportant role in the migration and retention of immunocompetent cellsin the CNS. The influence of E2 treatment on chemokine and chemokinereceptor mRNA in the spinal cords of WT and cytokine deficient mice wasmeasured using the RNAse protection assay (RPA). Total RNA was purifiedfrom spinal cords collected from mice at the peak of EAE (day 12-16post-immunization) and chemokine/chemokine receptor specific mRNA wasdetected using radiolabeled riboprobes. mRNAs coding for many of thechemokine and receptor family members were detectable in the spinalcords of WT C57BL/6 mice with EAE (FIG. 18). RANTES and IP-10 wereexpressed at the highest levels, followed by MIP-1α, MIP-2 and MCP-1.The levels of TCA-3 mRNA were below the limits of detection for thisassay. CCR5 was the most abundant chemokine receptor, followed by CCR1and CCR2 (FIG. 18), whereas CCR1b, CCR3, and CCR4 were below the levelof detection.

The expression of chemokine and chemokine receptor mRNA in cytokinedeficient mice with EAE was often markedly different from that in WTmice (FIG. 18). IL-4 deficient mice had reduced expression of RANTES andMIP-1α, but increased expression of MCP-1, whereas IL-10 and IFN-γdeficient mice had reduced expression of all chemokines tested exceptMCP-1. Of interest, TCA-3 mRNA was only detectable in INF-γ deficientmice. The expression of CCR1, CCR2, and CCR5 was nearly absent in IL-10deficient mice, but was only moderately altered in IL-4 and IFN-γdeficient mice. Thus, although distinct variations in the pattern ofchemokine or chemokine receptor expression occurred in the differentcytokine knockout mice, the development of EAE was not significantlychanged. These preliminary data provide evidence of the complexinteractions between chemokines and cytokines.

The expression of all chemokine and chemokine receptor mRNA wassignificantly diminished or absent in both WT and cytokine deficientmice treated with E2 (FIG. 18). This effect is likely the result of anE2-dependent decrease in the trafficking of inflammatory cells into theCNS, and possibly to its ability to inhibit the production of keyinflammatory factors.

Estrogen treatment reduced cytokine production in the CNS. Theexpression of cytokine mRNA in the spinal cords of mice at the peak ofEAE (day 12-16 post-immunization) was measured by RPA analysis.Messenger RNA encoding the pro-inflammatory cytokines IFN-γ, TNF-α, andLT-β were the most abundant (FIG. 19A). However, differences in theexpression level of cytokine mRNA were apparent in the cytokinedeficient mice. Messenger RNA for both TNF-α and IFN-γ weresubstantially lower in IL-10 knockout mice compared to WT, but nosignificant differences in LT-β levels were noted. Messenger RNA for allthree cytokines was profoundly lower in IFN-γ knockout mice compared toWT mice.

Surprisingly, low levels of IFN-γ mRNA were detected in IFN-γ deficientmice. These mice were created by homologous recombination of the firstexon (Dalton et al., Science 259:1739-1742 (1993), leaving the secondexon intact. Although it has not been reported previously, it ispossible that an mRNA product coding for the second exon is expressedand detected in our assay. Nevertheless, it is clear that lymphocytesfrom these mice fail to make a functional IFN-γ protein when measured byintracellular cytokine staining.

In all groups of mice, the levels of IL-4, IL-10, and TNF-β (LT-α) werebelow the limits of detection. The levels of LT-β, TNF-α, and IFN-γ mRNAin the spinal cords of E2 treated C57BL/6 and cytokine knockout micewere significantly reduced compared to untreated groups (FIG. 19A), withthe exception of LT-β levels in IFN-γ deficient mice.

Intracellular staining of CNS mononuclear cells with anti-cytokineantibodies was also performed. Mononuclear cells were recovered from thebrain and spinal cords of perfused mice at the peak of clinical disease.The total number of mononuclear cells recovered from the perfused CNS ofuntreated mice was 4-5 times higher than the number isolated from E2treated mice. These cells were stained with anti-CD4 cychrome labeledantibodies and the frequency of cytokine producing Vβ8.2+ helper T cellswas measured by staining with anti-Vβ8.2-FITC and PE-labeled cytokinespecific antibodies. As is shown in FIG. 19B, there was a dramaticreduction in the frequency and staining intensity (p<0.0001) of TNF-αand IFN-γ producing CD4+ T cells in the CNS of E2 treated mice. Based ontotal cell numbers recovered, E2 treatment caused a reduction ofpro-inflammatory cytokine producing CD4+ Vβ8.2+ T cells in CNS from29,000 cells/mouse to only 390 cells/mouse. A substantial reduction ofpro-inflammatory cytokine producing CD4+, Vβ8.2− T cells was alsoobserved (FIG. 19B). Taken together, these data confirm the RPA datapresented above, and directly support the hypothesis that E2 treatmentinhibits the activation and infiltration of pro-inflammatory cells intothe CNS.

Estrogen treatment failed to alter T cell proliferation and theexpression of cell surface adhesion and activation antigens.Proliferation of draining lymph node (LN) cells from either untreated orE2 treated mice was measured to determine if E2 could alter the abilityof T lymphocytes to recognize and respond to the immunizing antigen. LNcells were isolated from three representative mice for each group andthe cells pooled prior to stimulation with MOG 35-55 for 72 hr. Theresults shown in FIG. 20 illustrate that there was no effect of E2treatment on the LN proliferation response to MOG 35-55 in WT andcytokine deficient mice. Similarly, E2 treatment did not alter theresponse to antigen of splenocytes. These results indicate that E2treatment prevents the development of EAE without altering the abilityof MOG 35-55 specific T cells to proliferate in response to antigen.

The regulation of cell adhesion molecules is another possible mechanismby which estrogen treatment controls the migration of inflammatory cellsinto the CNS. The expression of cell surface adhesion andactivation/memory antigens was determined by staining withfluorochrome-labeled antibodies and flow cytometry. No significantdifferences in the expression of VLA-4, CD44, or CD62L were detectedbetween LN cells from E2 versus control mice with EAE (Table 18).Furthermore, no differences in activation markers (CD69, CD25, FASL,CD40L, CD28) were seen.

TABLE 18 % of Vβ8 · 2+/CD4 + T CELLS VLA-4 CD44hi CD69 FASL CD25 CD40LCD28 CD62lo untreated Exp #1 69.4 41.6 12.2 2.1  5.4 0 3.6 32.4 Exp #268.8 34.2 13.8 3.8 17.7 nd nd nd Exp #3 65.3 36.5 nd nd nd nd nd nd mean67.8 ± 2.2 37.4 ± 3.8 13.1 ± 1.1 3.0 ± 1.2 11.6 ± 8.7 E2 treated Exp #164.2 68.8 17.5 0.1  4.3 0 4.5 47.3 Exp #2 58.7 41.2 25.7 3.1 10.4 nd ndnd Exp #3 49.6 57.3 nd nd nd nd nd nd mean 57.5 ± 7.4 55.8 ± 13.8 21.6 ±5.8 1.6 ± 2.1  7.4 ± 4.3 P value  0.126  0.141  0.274 0.534  0.622

Estrogen treatment reduced the frequency of TNF-α secreting cells. Inorder to determine whether estrogen treatment promotes a shift towardsTh2 immunity, the frequency of both pro- and anti-inflammatory cytokineproducing cells in untreated and E2 treated mice was assessed, using theintracellular cytokine staining technique. Spleen cells were preparedfrom untreated and E2 treated mice at the peak of EAE (day 12-16post-immunization) and stimulated with MOG 35-55 for 24 hr, the last 6hr in the presence of Brefeldin A. The cells were stained with FITClabeled anti-Vβ8.1/8.2 TCR antibodies prior to fixation andpermeabilization, and then were stained with the indicated phycoerythrinlabeled anti-cytokine antibodies. Vβ8.1/8.2 TCR bearing T cells werefocused on because they are thought to comprise a major population ofthe MOG-35-55 specific T cell responses in H-2b mice.

The frequency of IFN-γ and TNF-α producing Vβ8.1/82 TCR+ cells wassimilar in untreated C57BL/6, as well as IL-4 and IL-10 deficient micewith EAE. However, the frequency of TNF-α producing cells wassignificantly lower in IFN-γ knockout mice, and as expected, there wereno detectable cells producing IFN-γ in these mice (FIGS. 21A-C). Thefrequency of TNF-α producing Vβ8.1/8.2 TCR+ cells was significantlydiminished in C57BL/6 mice (p=0.004), in IL-4 knockout mice (p=0.06) andin IL-10 knockout mice (p=0.001) but no further reduction in thefrequency of TNF-α producing cells was observed in E2 treated IFN-γknockout mice (FIG. 21C). The diminution in staining intensity of cellsfrom E2 treated mice also suggests that these cells also produce lowerlevels of TNF-α compared to the untreated mice. Since the number ofVβ8.1/8.2+ splenocytes recovered from the intact and cytokine knockoutmice was quite similar, it can be concluded that the total number ofTNF-α producing, MOG-reactive lymphocytes in the spleens of E2 treatedmice was significantly reduced. The frequency of Vβ8.2− cells producingTNF-α was also reduced in all of the E2-treated mouse groups, suggestingthat estrogen may influence cytokine production by encephalitogenic orrecruited T cells expressing different V genes, as well as otherinflammatory cells including macrophages.

The frequency of cells producing IFN-γ, IL-4, IL-10 and IL-12 was alsomeasured. Although there was a trend for E2 treated mice to have a lowerfrequency of IFN-γ producing cells (FIGS. 212B and C), these valuesfailed to attain statistical significance (p>0.05). Furthermore, thefrequency of IL-4, IL-10 and IL-12 producing cells was always below thelimits of detection for this assay. The failure to detect IL-4 and IL-10reactive cells suggests that E2 treatment did not significantly shiftthe cytokine response towards Th2 production.

EAE was suppressed in TNF-α deficient mice. The data presented aboveimplicate TNF-α producing cells as probable contributors to induction ofEAE. In order to further evaluate the pathogenic contribution of TNF-αin this model, the severity of EAE was compared in TNF-α deficient andWT control mice. Severe EAE developed in the majority of WT mice afterimmunization with MOG-35-55 peptide (Table 19). However, the incidenceand severity of EAE in TNF-α deficient mice was greatly diminished. Notonly did fewer mice develop disease, but the mean peak disease score andthe cumulative disease index were also profoundly reduced (Table 19).These data demonstrate that TNF-α producing cells are major contributorsto EAE induction, and their regulation by E2 provides an important newinsight into the regulatory effects of estrogen.

TABLE 19 Incidence Onset Mortality Peak CDI BL6.129s 8/8 11.8 ± 3.1 3/84.9 ± 1.5 78.3 ± 35.8 TNF-α 4/7 13.0 ± 0.8 0/7 0.6 ± 0.6 2.6 ± 4.6 KO Pvalue 0.153* 0.340 0.244 <0.0001 <0.0001

All journal article, reference and patent citations provided above, inparentheses or otherwise, are incorporated herein by reference in theirentirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention.

1. A method of ameliorating multiple sclerosis in a human subject,comprising administering a dose of estrogen to said human subjectsufficient to raise the serum concentration of estrogen in said human towithin the range from 30 pg/ml to 1000 pg/ml and administering atherapeutically effective amount of a T cell receptor peptide comprisinga human Vβ2, Vβ5.1, Vβ5.2, Vβ6.1, Vβ6.5, Vβ7, or a Vβ13 T cell receptorpeptide CDR2 region, or an analog of a Vβ5.2 T cell receptor peptidecomprising the amino acid sequence set forth as SEQ ID NO: 3, to saidhuman subject thereby ameliorating multiple sclerosis in said humansubject.
 2. The method of claim 1, wherein said human subject is female.3. The method of claim 1, wherein said human subject is male.
 4. Themethod of claim 1, wherein said estrogen is selected from the groupconsisting of 17β-estradiol, estriol and estrone.
 5. The method of claim4, wherein said estrogen is 17β-estradiol.
 6. The method of claim 1,wherein said dose of estrogen is an amount sufficient to raise the serumconcentration of estrogen in said human to within the range from 50pg/ml to 500 pg/ml.
 7. The method of claim 1, wherein said dose ofestrogen is an amount sufficient to raise the serum concentration ofestrogen to within the range from 100 pg/ml to 250 pg/ml.
 8. The methodof claim 1, wherein said estrogen is administered by a route selectedfrom oral, transdermal, respiratory, subcutaneous and intravenousroutes.
 9. The method of claim 1, wherein said amelioration is apparentby magnetic resonance imaging.
 10. The method of claim 1, wherein the Tcell receptor peptide comprises a Vβ CDR2 region comprising a Vβ2,Vβ5.1, Vβ5.2, Vβ6.1, Vβ6.5, Vβ7, or a Vβ13 amino acid sequence set forthin Table
 2. 11. The method of claim 1, wherein the T cell receptorpeptide comprises a Vβ5.2, Vβ6.5 or Vβ13 CDR2 region.
 12. The method ofclaim 10, wherein the T cell receptor peptide comprises a Vβ5.2, Vβ6.5or Vβ13 CDR2 region.
 13. The method of claim 10, wherein the T cellreceptor peptide comprises a Vβ5.2 CDR2 region.
 14. The method of claim1, wherein Vβ CDR2 region consists of a Vβ2 amino acid sequence setforth as one of SEQ ID NOs: 11-14; a Vβ5.1 amino acid sequence set forthas one of SEQ ID NO: 17 or SEQ ID NO:18; a Vβ5.2 amino acid sequence setforth as one of SEQ ID NOs: 2-3 or SEQ ID NO: 19, a Vβ6.1 amino acidsequence set forth as one of SEQ ID NO: 4, SEQ ID NO: 24 or SEQ ID NO:25, a Vβ6.5 amino acid sequence set forth as one of SEQ ID NO: 5 or SEQID NO: 30; a Vβ7 amino acid sequence set forth as one of SEQ ID NOs:33-55; or a Vβ13 amino acid sequence set forth as one of SEQ ID NO: 6 orSEQ ID NOs: 47-53.
 15. A method of ameliorating multiple sclerosis in ahuman subject, comprising administering a dose of estrogen to said humansubject sufficient to raise the serum concentration of estrogen in saidhuman to within the range from 30 pg/ml to 1000 pg/ml and administeringa therapeutically effective amount of a T cell receptor peptidecomprising a Vβ5.2 CDR2 region, a therapeutically effective amount of aT cell receptor peptide comprising Vβ6.5 CDR2 region, and atherapeutically effective amount of a T cell receptor peptide comprisinga Vβ13 CDR2 region, to said human subject thereby ameliorating multiplesclerosis in said human subject.
 16. The method of claim 11, wherein theVβ5.2 CDR2 region consists of the amino acid sequence set forth as oneof SEQ ID NOs: 2-3 or SEQ ID NO: 19; the Vβ6.5 CDR2 region consists ofthe amino acid sequence set forth as one of SEQ ID NO: 5 or SEQ ID NO:30; and the Vβ13 CDR2 region consists of the amino acid sequence setforth as one of SEQ ID NO: 6 or SEQ ID NOs: 47-53.
 17. The method ofclaim 11, wherein the Vβ5.2 CDR2 region consists of one of the aminoacid sequence set forth as one of SEQ ID NO: 2 or SEQ ID NO: 3; theVβ6.5 CDR2 region consists of the amino acid sequence set forth as SEQID NO: 5; and the Vβ13 CDR2 region consists of the amino acid sequenceset forth as SEQ ID NO:
 6. 18. The method of claim 15, wherein the Vβ5.2CDR2 region consists of one of the amino acid sequence set forth as oneof SEQ ID NO: 2 or SEQ ID NO: 3; the Vβ6.5 CDR2 region consists of theamino acid sequence set forth as SEQ ID NO: 5; and the Vβ13 CDR2 regionconsists of the amino acid sequence set forth as SEQ ID NO: 6.